Sex differences in Mbikael Bhousatedtv ior and metabolism Ivana Maric Department of Metabolic Physiology Institute of Neuroscience and Physiology Sahlgrenska Academy, University of Gothenburg Gothenburg, Sweden To my family Sex differences in behavior and metabolism © Ivana Maric 2023 ivana.maric@gu.se ISBN 978-91-8069-509-1 (PRINT) ISBN 978-91-8069-510-7 (PDF) AN ENM ENM Ä ÄR V R K VA N KEE Printed in Borås, Sweden 2023 Printed by Stema Specialtryck AB Trycksak3T0r4y1c k0s2a3k43041 0234 SS TT To my family Sex differences in behavior and metabolism © Ivana Maric 2023 ivana.maric@gu.se ISBN 978-91-8069-509-1 (PRINT) ISBN 978-91-8069-510-7 (PDF) Printed in Borås, Sweden 2023 Printed by Stema Specialtryck AB Sex differences in behavior and metabolism Ivana Maric Department of Metabolic Physiology, Institute of Neuroscience and Physiology Sahlgrenska Academy, University of Gothenburg, Sweden ABSTRACT Men and women exhibit distinct illness patterns and disparate responses to pharmacotherapies. However, there is a scarcity of preclinical studies that systematically compare the sexes. The overarching aim of this thesis was to identify sex differences, and their underlying mechanisms, in metabolic and behavioral control within rodent models of obesity and anxiety. Specifically, we explored novel brain targets and mechanisms for the control of appetite, energy expenditure, and emotionality. In Paper I, we found that mice and rats subjected to diet-induced obesity responded with sexually divergent eating behavior and adaptations in energy expenditure. In Paper II, we showed that obesity reduced the expression of interleukin-6 (IL-6) in the brain of males only, and that the role of IL-6 in the parabrachial nucleus (PBN) is sexually dimorphic, such that it is only necessary for normal brown adipose tissue thermogenesis in males. In Paper III, we discovered that the locus coeruleus (LC) is a novel site for the behavioral effects of the hunger hormone ghrelin. We demonstrated that males have higher ghrelin receptor levels in the LC, and that there is a sex difference in response to ghrelin receptor activation and blockade, in regards to food motivation and anxiety-like behavior. Finally, in Paper IV, we investigated whether brain-produced estrogen plays a role in body weight regulation. We found a sexually dimorphic role of aromatase in the amygdala, such that it is only necessary for normal energy homeostasis and food motivation in females. Collectively, the work presented in this thesis underscores the significance of considering biological sex in the context of energy balance regulation and associated behaviors. These findings contribute to a broader conversation on addressing sex differences in human disease with the ultimate goal of enhancing the success of drug development. Keywords: sex differences, eating, brown adipose tissue, motivation, anxiety-like behavior ISBN 978-91-8069-509-1 (PRINT) ISBN 978-91-8069-510-7 (PDF) Sex differences in behavior and metabolism Ivana Maric Department of Metabolic Physiology, Institute of Neuroscience and Physiology Sahlgrenska Academy, University of Gothenburg, Sweden ABSTRACT Men and women exhibit distinct illness patterns and disparate responses to pharmacotherapies. However, there is a scarcity of preclinical studies that systematically compare the sexes. The overarching aim of this thesis was to identify sex differences, and their underlying mechanisms, in metabolic and behavioral control within rodent models of obesity and anxiety. Specifically, we explored novel brain targets and mechanisms for the control of appetite, energy expenditure, and emotionality. In Paper I, we found that mice and rats subjected to diet-induced obesity responded with sexually divergent eating behavior and adaptations in energy expenditure. In Paper II, we showed that obesity reduced the expression of interleukin-6 (IL-6) in the brain of males only, and that the role of IL-6 in the parabrachial nucleus (PBN) is sexually dimorphic, such that it is only necessary for normal brown adipose tissue thermogenesis in males. In Paper III, we discovered that the locus coeruleus (LC) is a novel site for the behavioral effects of the hunger hormone ghrelin. We demonstrated that males have higher ghrelin receptor levels in the LC, and that there is a sex difference in response to ghrelin receptor activation and blockade, in regards to food motivation and anxiety-like behavior. Finally, in Paper IV, we investigated whether brain-produced estrogen plays a role in body weight regulation. We found a sexually dimorphic role of aromatase in the amygdala, such that it is only necessary for normal energy homeostasis and food motivation in females. Collectively, the work presented in this thesis underscores the significance of considering biological sex in the context of energy balance regulation and associated behaviors. These findings contribute to a broader conversation on addressing sex differences in human disease with the ultimate goal of enhancing the success of drug development. Keywords: sex differences, eating, brown adipose tissue, motivation, anxiety-like behavior ISBN 978-91-8069-509-1 (PRINT) ISBN 978-91-8069-510-7 (PDF) Sammanfattning på svenska fetmaläkemedel som riktar sig mot signalvägar relaterade till IL-6 bör beakta potentiella könsskillnader. Trots att sjukdomsförekomst och läkemedelsreaktioner skiljer sig mellan kvinnor och män baseras merparten av vår kunskap om medicin på forskning som har I vår tredje studie undersökte vi hormonet grelin som frisätts från magen och utförts på män och handjur. Denna snedvridning har gett upphov till spelar en avgörande roll för att överföra hungersignaler till hjärnan och påverka kunskapsluckor som bland annat har resulterat i att kvinnor drabbas av humöret. Vi upptäckte ett nytt hjärnområde där grelin verkar. Detta hjärnområde, läkemedelsbiverkningar i större utsträckning än män. Konsekvenserna sträcker känt som locus coeruleus (LC), är viktigt för kontrollen av motivation och sig bortom biverkningar – om grundläggande forskning inte undersöker emotionella tillstånd. Våra resultat visade att hanar och honor reagerar olika på eventuella könsbetingade skillnader kan vi gå miste om behandlingsmöjligheter grelinsignaler i LC. Grelin som administrerades till LC ökade matintaget och som hade varit gynnsamma för ett av könen. motivationen för socker hos båda könen, men minskade ångestliknande beteende endast hos honor. Att blockera grelinsignaler i samma hjärnområde minskade Denna avhandling utforskar könsskillnader i beteende och metabolism hos råttor matintaget och ökade ångesten hos båda könen, men påverkade motivation och möss. Vi har fokuserat på två vanliga hälsoproblem hos människor: fetma endast hos honor. och ångest. Fetma är en global hälsoutmaning kopplad till psykiatriska sjukdomar och nedsatt immunförsvar. Vår forskning visade att hanar och honor reagerar Avslutningsvis, i den fjärde studien, undersökte vi effekterna av aromatas, olika på fetma och övervikt. I vår första studie erbjöd vi möss och råttor av båda enzymet som är ansvarigt för syntesen av östrogen. Även om majoriteten av könen en diet med högt fett- och sockerinnehåll. Honor åt färre totala kalorier än östrogen produceras av könsorgan och fettvävnad, finns aromatas även i hjärnan hanar men föredrog den ohälsosamma dieten före sin vanliga diet. Detta tyder på hos både män och kvinnor. Trots det vet vi fortfarande lite om detta östrogen att deras matintag kan vara mer driven av njutning. Vi fann också att hanar och som produceras i hjärnan, särskilt med avseende på energibalans. När vi hämmade honor förbränner energi på olika sätt. Honråttor uppvisade större hjärnans aromatas, vilket minskade produktionen av östrogen, ökade endast energiförbränning, vilket troligen bidrog till större motståndskraftighet mot honor i vikt. Detta berodde på nedsatt energiförbrukning och ökat intag av viktuppgång. Å andra sidan var hanråttor bättre på att kompensera för det höga specifikt fettrik mat. Vi fann också att minskningen av aromatas i amygdala, en fettinnehållet i den ohälsosamma maten genom att öka aktiviteten i sin bruna hjärnregion som är involverad i emotionella processer, spelade en avgörande roll fettvävnad. Trots att den här kompensationen inte var tillräcklig för att förhindra i dessa effekter. Eftersom aromatashämmare redan används i klinisk praxis, viktuppgång så bidrog den troligen till att bibehålla normala blodsockernivåer. främst för behandling av bröstcancer, är det avgörande att förstå deras fulla påverkan på beteende och ämnesomsättning. I vår andra studie tittade vi på ämnet interleukin-6 (IL-6) som är känt för sin roll i inflammation. Vid fetma är nivåerna av IL-6 förhöjda i blodomloppet men Sammanfattningsvis betonar denna avhandling vikten av att inkludera både reducerade i den vätska som omger hjärnan, cerebrospinalvätskan. Vi observerade hanar och honor i prekliniska studier. Dessa resultat bidrar till en mer att hanråttor och möss med fetma hade reducerade nivåer IL-6 i en del av hjärnan omfattande diskussion om att systematiskt analysera eventuella könsskillnader i som kallas parabrachialkärnan (PBN), medan detta inte gällde för honor. Genom grundläggande forskning, med syftet att i slutändan förbättra framgången av att manipulera IL-6 i PBN kunde vi kontrollera kroppstemperatur och vikt hos läkemedelsutveckling. endast hanråttor. Intressant nog återställdes de reducerade nivåerna av IL-6 när de feta hanarna utsattes för kyla. Detta ledde också till en ökning av den bruna fettvävnadens förbränningsförmåga. Dessa resultat tyder på att utvecklingen av Sammanfattning på svenska fetmaläkemedel som riktar sig mot signalvägar relaterade till IL-6 bör beakta potentiella könsskillnader. Trots att sjukdomsförekomst och läkemedelsreaktioner skiljer sig mellan kvinnor och män baseras merparten av vår kunskap om medicin på forskning som har I vår tredje studie undersökte vi hormonet grelin som frisätts från magen och utförts på män och handjur. Denna snedvridning har gett upphov till spelar en avgörande roll för att överföra hungersignaler till hjärnan och påverka kunskapsluckor som bland annat har resulterat i att kvinnor drabbas av humöret. Vi upptäckte ett nytt hjärnområde där grelin verkar. Detta hjärnområde, läkemedelsbiverkningar i större utsträckning än män. Konsekvenserna sträcker känt som locus coeruleus (LC), är viktigt för kontrollen av motivation och sig bortom biverkningar – om grundläggande forskning inte undersöker emotionella tillstånd. Våra resultat visade att hanar och honor reagerar olika på eventuella könsbetingade skillnader kan vi gå miste om behandlingsmöjligheter grelinsignaler i LC. Grelin som administrerades till LC ökade matintaget och som hade varit gynnsamma för ett av könen. motivationen för socker hos båda könen, men minskade ångestliknande beteende endast hos honor. Att blockera grelinsignaler i samma hjärnområde minskade Denna avhandling utforskar könsskillnader i beteende och metabolism hos råttor matintaget och ökade ångesten hos båda könen, men påverkade motivation och möss. Vi har fokuserat på två vanliga hälsoproblem hos människor: fetma endast hos honor. och ångest. Fetma är en global hälsoutmaning kopplad till psykiatriska sjukdomar och nedsatt immunförsvar. Vår forskning visade att hanar och honor reagerar Avslutningsvis, i den fjärde studien, undersökte vi effekterna av aromatas, olika på fetma och övervikt. I vår första studie erbjöd vi möss och råttor av båda enzymet som är ansvarigt för syntesen av östrogen. Även om majoriteten av könen en diet med högt fett- och sockerinnehåll. Honor åt färre totala kalorier än östrogen produceras av könsorgan och fettvävnad, finns aromatas även i hjärnan hanar men föredrog den ohälsosamma dieten före sin vanliga diet. Detta tyder på hos både män och kvinnor. Trots det vet vi fortfarande lite om detta östrogen att deras matintag kan vara mer driven av njutning. Vi fann också att hanar och som produceras i hjärnan, särskilt med avseende på energibalans. När vi hämmade honor förbränner energi på olika sätt. Honråttor uppvisade större hjärnans aromatas, vilket minskade produktionen av östrogen, ökade endast energiförbränning, vilket troligen bidrog till större motståndskraftighet mot honor i vikt. Detta berodde på nedsatt energiförbrukning och ökat intag av viktuppgång. Å andra sidan var hanråttor bättre på att kompensera för det höga specifikt fettrik mat. Vi fann också att minskningen av aromatas i amygdala, en fettinnehållet i den ohälsosamma maten genom att öka aktiviteten i sin bruna hjärnregion som är involverad i emotionella processer, spelade en avgörande roll fettvävnad. Trots att den här kompensationen inte var tillräcklig för att förhindra i dessa effekter. Eftersom aromatashämmare redan används i klinisk praxis, viktuppgång så bidrog den troligen till att bibehålla normala blodsockernivåer. främst för behandling av bröstcancer, är det avgörande att förstå deras fulla påverkan på beteende och ämnesomsättning. I vår andra studie tittade vi på ämnet interleukin-6 (IL-6) som är känt för sin roll i inflammation. Vid fetma är nivåerna av IL-6 förhöjda i blodomloppet men Sammanfattningsvis betonar denna avhandling vikten av att inkludera både reducerade i den vätska som omger hjärnan, cerebrospinalvätskan. Vi observerade hanar och honor i prekliniska studier. Dessa resultat bidrar till en mer att hanråttor och möss med fetma hade reducerade nivåer IL-6 i en del av hjärnan omfattande diskussion om att systematiskt analysera eventuella könsskillnader i som kallas parabrachialkärnan (PBN), medan detta inte gällde för honor. Genom grundläggande forskning, med syftet att i slutändan förbättra framgången av att manipulera IL-6 i PBN kunde vi kontrollera kroppstemperatur och vikt hos läkemedelsutveckling. endast hanråttor. Intressant nog återställdes de reducerade nivåerna av IL-6 när de feta hanarna utsattes för kyla. Detta ledde också till en ökning av den bruna fettvävnadens förbränningsförmåga. Dessa resultat tyder på att utvecklingen av Научно популарни сажетак специфично у ПБЈ, успели смо да контролишемо телесну температуру и добитак телесне масе мужјака пацова. Занимљиво, излагање животиња хладноћи успоставило је нормалне нивое ИЛ-6 и повећало активност мрког Упркос разликама у учесталости обољења и реакцијама на лекове између масног ткива у сагоревању калорија. Ови резултати указују на то да будући мушкараца и жена, већина нашег медицинског знања заснива се на третмани гојазности морају узети у обзир полне разлике. истраживањима изведеним преваcxодно на мушкарцима и мужјацима. Ова полна пристрасност у истраживању довела је до непотпуног разумевања У трећем раду из ове дисертације испитивали смо хормон грелин који се начина на који терапија утиче на жене, што често доводи до испољавања ослобађа из црева и игра кључну улогу у преносу сигнала за глад ка мозгу, више нуспојава лекова код жена у односу на мушкарце. Међутим, полна такође утичући на расположење. Открили смо ново место испољавања пристрасност у истраживању има, поред нуспојава лекова, и друге дејства грелина. Тај регион мозга, познатији као ромбоидна јама (лоцус последице. Ако не узимамо у обзир оба пола и поређење између њих, цоерулеус , ЛЦ), укључен је у контролу мотивације и емотивних стања. можемо пропустити терапеутике који би били врло ефикасни код једног Наши резултати су показали да мужјаци и женке испољавају другачији пола али никад не би прошли клиничка испитивања због недостатка одговор на стимулацију грелином у ЛЦ. Примена грелина у ЛЦ повећала је статистичке анализе у односу на пол. унос хране и жељу за шећером у оба пола, али је смањила анксиозности- слично понашање само код женки. Блокада сигнала грелина у истом У овој докторској дисертацији смо истраживали разлике у метаболизму и региону мозга смањила је унос хране и повећала анксиозност код оба пола. понашању између мужјака и женки пацова и мишева. Усредсредили смо се Међутим, блокада сигнала грелина је утицала на искуство задовољства на два честа обољења код људи: гојазност и анксиозност. Гојазност је храном само код женки. глобални здравствени изазов који је повезан са психијатријским и имунским поремећајима. Наше истраживање открило је да мужјаци и женке различито Коначно, четврти рад из ове дисертације бави се ароматазом - ензимом одговарају на гојазност и прекомерно стицање телесне масе. У првом раду одговорним за настанак естрогена. Иако се већина естрогена производи у из ове дисертације, понудили смо мишевима и пацовима оба пола избор репродуктивним органима и масном ткиву, ароматаза је такође присутна и у између стандардне и дијете богате мастима и шећерима. Женке су уносиле мозгу оба пола. Међутим, о овом естрогену који настаје у мозгу се и даље мање калорија свеукупно у односу на мужјаке али су бирале нездраву храну врло мало зна, нарочито у односу на енергетски баланс. Када смо спречили у односу на нормалну. Ово указује да су навике у иcxрани женки можда више дејство ароматазе у мозгу, само су женке добиле на тежини. Овај добитак подстакнуте задовољством. Такође смо показали да мужјаци и женке телесне масе је био последица смањеног трошења енергије и повећаног различито сагоревају енергију. У почетним условима, женке пацова су уноса специфично калоричне хране. Такође смо открили да је смањење показале већу локомоторну активност и активност мрког масног ткива, што ароматазе у амигдали, региону мозга укљученом у емотивне процесе, је вероватно допринело почетном одолевању у добијању телесне масе. Са одиграло кључну улогу на поменуте процесе. Имајући у виду да су друге стране, мужјаци пацова су боље балансирали дијету богату мастима инхибитори ароматазе класа лекова који су већ у клиничкој употреби, тако што су појачавали метаболизам путем активације мрког масног ткива углавном за третман канцера дојке, изузетно је важно разумети њихов које помаже у сагоревању калорија и регулацији шећера у крви. свеукупан утицај на понашање и метаболизам. У другом раду из дисертације концентрисали смо се на супстанцу под Укратко, наши резултати истичу важност укључивања оба пола у пре именом интерлеукин-6 (ИЛ-6) која је позната по својој улози у запаљенским клиничка истраживања. Ови резултати доприносе ширем дијалогу о апсекту процесима. Док је гојазност повезана са високим нивоом ИЛ-6 у крви, нивои полних разлика у болестима људи са крајњим циљем повећавања ИЛ-6 у течности која окружује мозак, цереброспиналној течности, се успешности развоја нових лекова. смањују са гојазношћу. Приметили смо да гојазни мужјаци мишева и пацова имају смањени ниво ИЛ-6 у делу мозга који се зове парабрахијално једро (ПБЈ), што није био случај са гојазним женкама. Подешавањем нивоа ИЛ-6 Научно популарни сажетак специфично у ПБЈ, успели смо да контролишемо телесну температуру и добитак телесне масе мужјака пацова. Занимљиво, излагање животиња хладноћи успоставило је нормалне нивое ИЛ-6 и повећало активност мрког Упркос разликама у учесталости обољења и реакцијама на лекове између масног ткива у сагоревању калорија. Ови резултати указују на то да будући мушкараца и жена, већина нашег медицинског знања заснива се на третмани гојазности морају узети у обзир полне разлике. истраживањима изведеним преваcxодно на мушкарцима и мужјацима. Ова полна пристрасност у истраживању довела је до непотпуног разумевања У трећем раду из ове дисертације испитивали смо хормон грелин који се начина на који терапија утиче на жене, што често доводи до испољавања ослобађа из црева и игра кључну улогу у преносу сигнала за глад ка мозгу, више нуспојава лекова код жена у односу на мушкарце. Међутим, полна такође утичући на расположење. Открили смо ново место испољавања пристрасност у истраживању има, поред нуспојава лекова, и друге дејства грелина. Тај регион мозга, познатији као ромбоидна јама (лоцус последице. Ако не узимамо у обзир оба пола и поређење између њих, цоерулеус , ЛЦ), укључен је у контролу мотивације и емотивних стања. можемо пропустити терапеутике који би били врло ефикасни код једног Наши резултати су показали да мужјаци и женке испољавају другачији пола али никад не би прошли клиничка испитивања због недостатка одговор на стимулацију грелином у ЛЦ. Примена грелина у ЛЦ повећала је статистичке анализе у односу на пол. унос хране и жељу за шећером у оба пола, али је смањила анксиозности- слично понашање само код женки. Блокада сигнала грелина у истом У овој докторској дисертацији смо истраживали разлике у метаболизму и региону мозга смањила је унос хране и повећала анксиозност код оба пола. понашању између мужјака и женки пацова и мишева. Усредсредили смо се Међутим, блокада сигнала грелина је утицала на искуство задовољства на два честа обољења код људи: гојазност и анксиозност. Гојазност је храном само код женки. глобални здравствени изазов који је повезан са психијатријским и имунским поремећајима. Наше истраживање открило је да мужјаци и женке различито Коначно, четврти рад из ове дисертације бави се ароматазом - ензимом одговарају на гојазност и прекомерно стицање телесне масе. У првом раду одговорним за настанак естрогена. Иако се већина естрогена производи у из ове дисертације, понудили смо мишевима и пацовима оба пола избор репродуктивним органима и масном ткиву, ароматаза је такође присутна и у између стандардне и дијете богате мастима и шећерима. Женке су уносиле мозгу оба пола. Међутим, о овом естрогену који настаје у мозгу се и даље мање калорија свеукупно у односу на мужјаке али су бирале нездраву храну врло мало зна, нарочито у односу на енергетски баланс. Када смо спречили у односу на нормалну. Ово указује да су навике у иcxрани женки можда више дејство ароматазе у мозгу, само су женке добиле на тежини. Овај добитак подстакнуте задовољством. Такође смо показали да мужјаци и женке телесне масе је био последица смањеног трошења енергије и повећаног различито сагоревају енергију. У почетним условима, женке пацова су уноса специфично калоричне хране. Такође смо открили да је смањење показале већу локомоторну активност и активност мрког масног ткива, што ароматазе у амигдали, региону мозга укљученом у емотивне процесе, је вероватно допринело почетном одолевању у добијању телесне масе. Са одиграло кључну улогу на поменуте процесе. Имајући у виду да су друге стране, мужјаци пацова су боље балансирали дијету богату мастима инхибитори ароматазе класа лекова који су већ у клиничкој употреби, тако што су појачавали метаболизам путем активације мрког масног ткива углавном за третман канцера дојке, изузетно је важно разумети њихов које помаже у сагоревању калорија и регулацији шећера у крви. свеукупан утицај на понашање и метаболизам. У другом раду из дисертације концентрисали смо се на супстанцу под Укратко, наши резултати истичу важност укључивања оба пола у пре именом интерлеукин-6 (ИЛ-6) која је позната по својој улози у запаљенским клиничка истраживања. Ови резултати доприносе ширем дијалогу о апсекту процесима. Док је гојазност повезана са високим нивоом ИЛ-6 у крви, нивои полних разлика у болестима људи са крајњим циљем повећавања ИЛ-6 у течности која окружује мозак, цереброспиналној течности, се успешности развоја нових лекова. смањују са гојазношћу. Приметили смо да гојазни мужјаци мишева и пацова имају смањени ниво ИЛ-6 у делу мозга који се зове парабрахијално једро (ПБЈ), што није био случај са гојазним женкама. Подешавањем нивоа ИЛ-6 List of papers Scientific contributions beyond this thesis This thesis is based on the following studies, referred to in the text by their Peripherally restricted oxytocin is sufficient to reduce food intake and Roman numerals. motivation, while CNS entry is required for locomotor and taste avoidance effects. I. Sex and species differences in the development of diet-induced Asker M, Krieger JP, Liles A, Tinsley IC, Borner T, Maric I, Doebley S, Furst obesity and metabolic disturbances in rodents. CD, Börchers S, Longo F, Bhat YR, De Jonghe BC, Hayes MR, Doyle RP, Maric I, Krieger JP, van der Velden P, Börchers S, Asker M, Vujicic Skibicka KP. M, Wernstedt Asterholm I, Skibicka KP. Diabetes, Obesity and Metabolism, 2023; 25(3): 856-877. Frontiers in Nutrition, 2022; 9: 828522. Neural pathway for gut feelings: vagal interoceptive feedback from the II. Parabrachial interleukin-6 reduces body weight and food intake gastrointestinal tract is a critical modulator of anxiety-like behavior. and increases thermogenesis to regulate energy metabolism. Krieger JP, Asker M, van der Velden P, Börchers S, Richard JE, Maric I, Mishra D, Richard JE, Maric I, Porteiro B, Häring M, Kooijman S, Longo F, Singh A, de Lartigue G, Skibicka KP. Musovic S, Eerola K, López-Ferreras L, Peris E, Grycel K, Biological Psychiatry, 2022; 92(9): 709-721. Shevchouk OT, Micallef P, Olofsson CS, Wernstedt Asterholm I, Grill HJ, Nogueiras R, Skibicka KP. From an empty stomach to anxiolysis: molecular and behavioral Cell reports, 2019; 26(11): 3011-3026.e5. assessment of sex differences in the ghrelin axis of rats. Börchers S, Krieger JP, Maric I, Carl J, Abraham M, Longo F, Asker M, III. From the stomach to locus coeruleus: new neural substrate for Richard JE, Skibicka KP. ghrelin’s effects on ingestive, motivated and anxiety-like Frontiers in Endocrinology, 2022; 13: 901669. behaviors. Maric I, López-Ferreras L, Bhat Y, Asker M, Börchers S, Bellfy L, Commonly-used rodent tests of anxiety-like behavior lack predictive Byun S, Kwapis J, Skibicka KP. validity for human sex differences. Frontiers in Pharmacology, 2023; 14: 1286805. Börchers S, Krieger JP, Asker M, Maric I, Skibicka KP. Psychoneuroendocrinology, 2022; 141: 105733. IV. Sex-specific effects of amygdala aromatase in the control of energy balance and food reward. Maric I, Richard JE, Taing L, López-Ferreras L, Byun S, Bhat Y, Skibicka KP. Manuscript List of papers Scientific contributions beyond this thesis This thesis is based on the following studies, referred to in the text by their Peripherally restricted oxytocin is sufficient to reduce food intake and Roman numerals. motivation, while CNS entry is required for locomotor and taste avoidance effects. I. Sex and species differences in the development of diet-induced Asker M, Krieger JP, Liles A, Tinsley IC, Borner T, Maric I, Doebley S, Furst obesity and metabolic disturbances in rodents. CD, Börchers S, Longo F, Bhat YR, De Jonghe BC, Hayes MR, Doyle RP, Maric I, Krieger JP, van der Velden P, Börchers S, Asker M, Vujicic Skibicka KP. M, Wernstedt Asterholm I, Skibicka KP. Diabetes, Obesity and Metabolism, 2023; 25(3): 856-877. Frontiers in Nutrition, 2022; 9: 828522. Neural pathway for gut feelings: vagal interoceptive feedback from the II. Parabrachial interleukin-6 reduces body weight and food intake gastrointestinal tract is a critical modulator of anxiety-like behavior. and increases thermogenesis to regulate energy metabolism. Krieger JP, Asker M, van der Velden P, Börchers S, Richard JE, Maric I, Mishra D, Richard JE, Maric I, Porteiro B, Häring M, Kooijman S, Longo F, Singh A, de Lartigue G, Skibicka KP. Musovic S, Eerola K, López-Ferreras L, Peris E, Grycel K, Biological Psychiatry, 2022; 92(9): 709-721. Shevchouk OT, Micallef P, Olofsson CS, Wernstedt Asterholm I, Grill HJ, Nogueiras R, Skibicka KP. From an empty stomach to anxiolysis: molecular and behavioral Cell reports, 2019; 26(11): 3011-3026.e5. assessment of sex differences in the ghrelin axis of rats. Börchers S, Krieger JP, Maric I, Carl J, Abraham M, Longo F, Asker M, III. From the stomach to locus coeruleus: new neural substrate for Richard JE, Skibicka KP. ghrelin’s effects on ingestive, motivated and anxiety-like Frontiers in Endocrinology, 2022; 13: 901669. behaviors. Maric I, López-Ferreras L, Bhat Y, Asker M, Börchers S, Bellfy L, Commonly-used rodent tests of anxiety-like behavior lack predictive Byun S, Kwapis J, Skibicka KP. validity for human sex differences. Frontiers in Pharmacology, 2023; 14: 1286805. Börchers S, Krieger JP, Asker M, Maric I, Skibicka KP. Psychoneuroendocrinology, 2022; 141: 105733. IV. Sex-specific effects of amygdala aromatase in the control of energy balance and food reward. Maric I, Richard JE, Taing L, López-Ferreras L, Byun S, Bhat Y, Skibicka KP. Manuscript CONTENT Abbreviations INTRODUCTION ......................................................................................... 1 ORIGINS OF SEX DIFFERENCES ...................................................................... 2 AAV adeno-associated virus PERIPHERAL SIGNALS IN ENERGY BALANCE REGULATION ............................. 5 Ghrelin ........................................................................................................ 7 AgRP agouti-gene-related protein Interleukin-6 ................................................................................................ 8 CENTRAL NERVOUS SYSTEM INTEGRATION .................................................... 9 AR androgen receptor The hypothalamus ........................................................................................ 10 Arc arcuate nucleus of the hypothalamus The hindbrain ............................................................................................. 10 The reward system ........................................................................................ 12 ASR acoustic startle response THE SEXUAL DIMORPHISM OF ADIPOSITY ..................................................... 14 THE SEX BIAS IN BIOMEDICAL RESEARCH ..................................................... 16 BAT brown adipose tissue AIM ............................................................................................................... 18 CART cocaine- and amphetamine-regulated transcript METHODOLOGICAL CONSIDERATIONS .......................................... 19 CCK cholecystokinin EXPERIMENTAL MODEL ............................................................................... 19 CNS central nervous system OBESOGENIC DIETS ..................................................................................... 20 SURGERIES .................................................................................................. 21 CRH corticotropin-releasing hormone VIRAL VECTORS ........................................................................................... 22 DRUGS ......................................................................................................... 23 CSF cerebrospinal fluid TEMPERATURE AND LOCOMOTOR ACTIVITY MEASUREMENTS ...................... 24 ER estrogen receptor OPERANT CONDITIONING ........................................................................... 25 ANXIETY-LIKE BEHAVIOR ............................................................................ 26 FISH fluorescent in situ hybridization BIOCHEMICAL PROCEDURES ........................................................................ 27 S ................................................................................. 29 GHSR growth hormone secretagogue receptor, ghrelin receptor TATISTICAL ANALYSIS RESULTS ..................................................................................................... 30 GLP-1 glucagon-like peptide 1 PAPER I ....................................................................................................... 30 HPA hypothalamic-pituitary-adrenal PAPER II ...................................................................................................... 31 PAPER III ..................................................................................................... 33 IHC immunohistochemistry PAPER IV ..................................................................................................... 34 IL-6 interleukin-6 DISCUSSION ............................................................................................... 36 LC locus coeruleus ACKNOWLEDGEMENTS ........................................................................ 42 LEAP2 liver-enriched antimicrobial peptide-2 REFERENCES ............................................................................................. 44 CONTENT Abbreviations INTRODUCTION ......................................................................................... 1 ORIGINS OF SEX DIFFERENCES ...................................................................... 2 AAV adeno-associated virus PERIPHERAL SIGNALS IN ENERGY BALANCE REGULATION ............................. 5 Ghrelin ........................................................................................................ 7 AgRP agouti-gene-related protein Interleukin-6 ................................................................................................ 8 CENTRAL NERVOUS SYSTEM INTEGRATION .................................................... 9 AR androgen receptor The hypothalamus ........................................................................................ 10 Arc arcuate nucleus of the hypothalamus The hindbrain ............................................................................................. 10 The reward system ........................................................................................ 12 ASR acoustic startle response THE SEXUAL DIMORPHISM OF ADIPOSITY ..................................................... 14 THE SEX BIAS IN BIOMEDICAL RESEARCH ..................................................... 16 BAT brown adipose tissue AIM ............................................................................................................... 18 CART cocaine- and amphetamine-regulated transcript METHODOLOGICAL CONSIDERATIONS .......................................... 19 CCK cholecystokinin EXPERIMENTAL MODEL ............................................................................... 19 CNS central nervous system OBESOGENIC DIETS ..................................................................................... 20 SURGERIES .................................................................................................. 21 CRH corticotropin-releasing hormone VIRAL VECTORS ........................................................................................... 22 DRUGS ......................................................................................................... 23 CSF cerebrospinal fluid TEMPERATURE AND LOCOMOTOR ACTIVITY MEASUREMENTS ...................... 24 ER estrogen receptor OPERANT CONDITIONING ........................................................................... 25 ANXIETY-LIKE BEHAVIOR ............................................................................ 26 FISH fluorescent in situ hybridization BIOCHEMICAL PROCEDURES ........................................................................ 27 S ................................................................................. 29 GHSR growth hormone secretagogue receptor, ghrelin receptor TATISTICAL ANALYSIS RESULTS ..................................................................................................... 30 GLP-1 glucagon-like peptide 1 PAPER I ....................................................................................................... 30 HPA hypothalamic-pituitary-adrenal PAPER II ...................................................................................................... 31 PAPER III ..................................................................................................... 33 IHC immunohistochemistry PAPER IV ..................................................................................................... 34 IL-6 interleukin-6 DISCUSSION ............................................................................................... 36 LC locus coeruleus ACKNOWLEDGEMENTS ........................................................................ 42 LEAP2 liver-enriched antimicrobial peptide-2 REFERENCES ............................................................................................. 44 LH lateral hypothalamus Definitions in short MCH melanin-concentrating hormone Sex differences are defined as biological differences determined from the mRNA messenger RNA gonadal and chromosomal composition. While this thesis categorizes sex as NAc nucleus accumbens male or female in the context of experimental rodents, it is important to recognize that in animals (including humans) variations to this binarity do NPY neuropeptide Y occur. NTS nucleus tractus solitarus Gender differences are relevant only for research with humans and OF open field encompass gender identity, social structures and culturally acquired or ORX orexin attributed characteristics. OT oxytocin PBN parabrachial nucleus PFC prefrontal cortex POMC proopiomelanocortin PR progestin receptor PVH paraventricular nucleus PYY peptide YY qPCR quantitative polymerase chain reaction siRNA small interfering RNA TH tyrosine hydroxylase UCP-1 uncoupling protein-1 VMH ventromedial hypothalamic area VTA ventral tegmental area WAT white adipose tissue LH lateral hypothalamus Definitions in short MCH melanin-concentrating hormone Sex differences are defined as biological differences determined from the mRNA messenger RNA gonadal and chromosomal composition. While this thesis categorizes sex as NAc nucleus accumbens male or female in the context of experimental rodents, it is important to recognize that in animals (including humans) variations to this binarity do NPY neuropeptide Y occur. NTS nucleus tractus solitarus Gender differences are relevant only for research with humans and OF open field encompass gender identity, social structures and culturally acquired or ORX orexin attributed characteristics. OT oxytocin PBN parabrachial nucleus PFC prefrontal cortex POMC proopiomelanocortin PR progestin receptor PVH paraventricular nucleus PYY peptide YY qPCR quantitative polymerase chain reaction siRNA small interfering RNA TH tyrosine hydroxylase UCP-1 uncoupling protein-1 VMH ventromedial hypothalamic area VTA ventral tegmental area WAT white adipose tissue Ivana Maric Ivana Maric balance point of view, it may not be possible to solve the problem of INTRODUCTION excessive weight gain by focusing on eating behavior alone. Energy expenditure is divided in three main components: basal metabolism, Sex differences in prevalence, manifestation, and drug response have adaptive thermogenesis and physical activity. On this side of the energy become evident in numerous medical conditions (Mauvais-Jarvis et al., balance equation, brown fat stands out as an intriguing therapeutic 2020). While these disparities are increasingly recognized, the target due to its inherent ability to burn calories (Nedergaard & foundation of our knowledge about health and disease is Cannon, 2010). Nonetheless, our understanding of the tissue's role in predominantly derived from research conducted on male cells, male the development and prevention of obesity remains limited. rodents, and men (Beery & Zucker, 2011; Woitowich et al., 2020). This bias holds significant implications, no less in the context of one of the The consequences of excessive weight gain extend far beyond most pressing global health challenges of our time – the striking surge metabolic concerns; it intertwines with psychiatric disorders and in overweight and obesity rates. Women are disproportionately impaired immune function (Bapat et al., 2022; Milaneschi et al., 2019). affected by eating disorders and morbid obesity compared to men To combat the rise of obesity and comorbidities effectively, a (Flegal et al., 2016). Differences between genders can arise from the comprehensive understanding of the neural circuits governing energy sociocultural factors imposed on humans, however, animal studies intake and expenditure is imperative. Despite the increasing have revealed that there is an effect of biological sex in many aspects recognition of the interaction between sex and body weight regulation, of energy balance regulation (Benz et al., 2012; Morselli et al., 2016). further explorations about how the underlying mechanisms differ Some of these, albeit not all, can be attributed sex hormones and their between males and females are necessary. This thesis aims to fluctuations throughout life (Wang & Xu, 2019). encourage a deeper appreciation for sex as a modifier of behavior and metabolism, as part of a crucial step toward research that will bring Weight gain is a result of a positive energy balance, where energy intake advantages to men and women alike. exceeds energy expenditure. Appetite is complex and modulated by the body's internal state and the perception and processing of Origins of sex differences environmental stimuli. In the control of eating, the brain integrates an array of peripheral signals communicating energy reserves, along with Biological sex differences result from the interplay of genetic and intricate signals encompassing sensory pleasures, past experiences, endocrine mechanisms that evolved due to evolutionary forces. Some emotional states, circadian rhythms, immune responses, and more. The are intrinsic and driven by direct effects of sex chromosome genes, the role of food in modern Western society has transcended its organizational effects of hormones during early development, and fundamental purpose of providing nutrition for survival. The abundant epigenetic chromatic modifications. The initiation of biological sex availability of tasty, energy-dense food, promotes overeating and leads differences is controlled by genes on the X and Y sex chromosomes, to an excess accumulation of body fat. In our battle against obesity, with females typically possessing the XX karyotype, and males the XY gaining insights into the fundamental mechanisms driving this karyotype. During development, male and female embryos are exposed disrupted eating behavior is of utmost importance. From an energy to distinct surges of androgens and estrogens respectively (Arnold & 1 2 Ivana Maric Ivana Maric balance point of view, it may not be possible to solve the problem of INTRODUCTION excessive weight gain by focusing on eating behavior alone. Energy expenditure is divided in three main components: basal metabolism, Sex differences in prevalence, manifestation, and drug response have adaptive thermogenesis and physical activity. On this side of the energy become evident in numerous medical conditions (Mauvais-Jarvis et al., balance equation, brown fat stands out as an intriguing therapeutic 2020). While these disparities are increasingly recognized, the target due to its inherent ability to burn calories (Nedergaard & foundation of our knowledge about health and disease is Cannon, 2010). Nonetheless, our understanding of the tissue's role in predominantly derived from research conducted on male cells, male the development and prevention of obesity remains limited. rodents, and men (Beery & Zucker, 2011; Woitowich et al., 2020). This bias holds significant implications, no less in the context of one of the The consequences of excessive weight gain extend far beyond most pressing global health challenges of our time – the striking surge metabolic concerns; it intertwines with psychiatric disorders and in overweight and obesity rates. Women are disproportionately impaired immune function (Bapat et al., 2022; Milaneschi et al., 2019). affected by eating disorders and morbid obesity compared to men To combat the rise of obesity and comorbidities effectively, a (Flegal et al., 2016). Differences between genders can arise from the comprehensive understanding of the neural circuits governing energy sociocultural factors imposed on humans, however, animal studies intake and expenditure is imperative. Despite the increasing have revealed that there is an effect of biological sex in many aspects recognition of the interaction between sex and body weight regulation, of energy balance regulation (Benz et al., 2012; Morselli et al., 2016). further explorations about how the underlying mechanisms differ Some of these, albeit not all, can be attributed sex hormones and their between males and females are necessary. This thesis aims to fluctuations throughout life (Wang & Xu, 2019). encourage a deeper appreciation for sex as a modifier of behavior and metabolism, as part of a crucial step toward research that will bring Weight gain is a result of a positive energy balance, where energy intake advantages to men and women alike. exceeds energy expenditure. Appetite is complex and modulated by the body's internal state and the perception and processing of Origins of sex differences environmental stimuli. In the control of eating, the brain integrates an array of peripheral signals communicating energy reserves, along with Biological sex differences result from the interplay of genetic and intricate signals encompassing sensory pleasures, past experiences, endocrine mechanisms that evolved due to evolutionary forces. Some emotional states, circadian rhythms, immune responses, and more. The are intrinsic and driven by direct effects of sex chromosome genes, the role of food in modern Western society has transcended its organizational effects of hormones during early development, and fundamental purpose of providing nutrition for survival. The abundant epigenetic chromatic modifications. The initiation of biological sex availability of tasty, energy-dense food, promotes overeating and leads differences is controlled by genes on the X and Y sex chromosomes, to an excess accumulation of body fat. In our battle against obesity, with females typically possessing the XX karyotype, and males the XY gaining insights into the fundamental mechanisms driving this karyotype. During development, male and female embryos are exposed disrupted eating behavior is of utmost importance. From an energy to distinct surges of androgens and estrogens respectively (Arnold & 1 2 Ivana Maric Ivana Maric Breedlove, 1985). This leads to ‘organizational effects’, which have spans slightly shorter than a month on average, while the rodent permanent impact on shaping anatomy and physiology. For instance, estrous cycle is approximately four days long. Nonetheless, the male rodents and humans respond to weight loss by compensatory hormonal fluctuations are very similar between species and affect, and overeating, while females compensate by reduced energy expenditure shape, brain physiology (Rocks et al., 2022). In the past, the exclusion (Shi et al., 2007; Valle et al., 2005; Zandian et al., 2011). This outcome of female animals in pre-clinical research was often justified with the was suggested to be due to the masculinization of the developing brain, misconception that females exhibited more variable behavior than resulting in sex-specific morphological differences within the males due to these hormonal fluctuations. However, recent research hypothalamic melanocortin system in mice (Nohara et al., 2011). has dispelled this myth, as it has been shown that male rodents are Beyond the organizational effects, a big portion of observed sex equally variable and also undergo hormonal changes, albeit not in the differences are due to ‘activational effects’ that are mediated through predictable cyclical pattern observed in females (Becker et al., 2016; the acute effects of sex steroids (Frye et al., 2008; Santollo & Eckel, Prendergast et al., 2014; Smarr & Kriegsfeld, 2022). 2008; Walf & Frye, 2010). The effects of ovarian estrogens have been extensively studied in the Sex steroids encompass estrogens, androgens and progestins. These context of eating behavior. During the ovarian cycle, variations in food hormones interact with their specific receptors, namely progestin intake occur in both women and rodents. Lowest food intake is receptors (PR), androgen receptors (AR) and estrogen receptors (ER), observed following periods of high estradiol levels, namely the distributed throughout the body and the brain of both males and periovulatory phase in women and estrus in rats (Danker-Hopfe et al., females (MacLusky & McEwen, 1980; Shughrue et al., 1997; Simerly et 1995; Roney & Simmons, 2017). The anorexic effects of estradiol are al., 1990). Progestins are synthesized from cholesterol, and can be further confirmed with ovariectomy (Asarian & Geary, 2002). In rats, further converted to androgens (Miller & Auchus, 2011). Subsequently, the surgical removal of ovaries results in hyperphagia (overeating), estrogens are synthesized from androgens by the enzyme aromatase increased food reward, and body weight gain. Central administration (encoded by the CYP19A1 gene) (Simpson et al., 1994). Both males of estradiol is enough to reverse these effects, and ERs in the brain are and females produce testosterone and estrogen. In men, testes are necessary for estradiols anorexigenic effects (Palmer & Gray, 1986; producing majority of circulating androgens, while the same holds true Rivera & Eckel, 2010). The influence of androgens on eating behavior for ovaries and estrogen in premenopausal women. In contrast, in men in males have received less attention, possibly because androgen levels and postmenopausal women estrogen synthesis primarily takes place remain relatively stable throughout reproductive life and are not in other tissues than the gonads. This is possible due to the presence subject to abrupt changes as the ones associated with pregnancy or of aromatase in organs beyond the gonads, including the adipose tissue menopause (Kaufman & Vermeulen, 2005). Hypogonadism in men is and brain (Callard et al., 1978; McTernan et al., 2000; Roselli et al., associated with obesity, but castration leads to weight loss which is 1998). partially reversed with testosterone administration (Fernandez et al., 2019; Gentry & Wade, 1976; Kim et al., 2021). Notably, the Women, as well as female rodents, have fluctuating plasma levels of interpretations of testosterones effects can be complicated by sex hormones produced by the ovaries. The human menstrual cycle 3 4 Ivana Maric Ivana Maric Breedlove, 1985). This leads to ‘organizational effects’, which have spans slightly shorter than a month on average, while the rodent permanent impact on shaping anatomy and physiology. For instance, estrous cycle is approximately four days long. Nonetheless, the male rodents and humans respond to weight loss by compensatory hormonal fluctuations are very similar between species and affect, and overeating, while females compensate by reduced energy expenditure shape, brain physiology (Rocks et al., 2022). In the past, the exclusion (Shi et al., 2007; Valle et al., 2005; Zandian et al., 2011). This outcome of female animals in pre-clinical research was often justified with the was suggested to be due to the masculinization of the developing brain, misconception that females exhibited more variable behavior than resulting in sex-specific morphological differences within the males due to these hormonal fluctuations. However, recent research hypothalamic melanocortin system in mice (Nohara et al., 2011). has dispelled this myth, as it has been shown that male rodents are Beyond the organizational effects, a big portion of observed sex equally variable and also undergo hormonal changes, albeit not in the differences are due to ‘activational effects’ that are mediated through predictable cyclical pattern observed in females (Becker et al., 2016; the acute effects of sex steroids (Frye et al., 2008; Santollo & Eckel, Prendergast et al., 2014; Smarr & Kriegsfeld, 2022). 2008; Walf & Frye, 2010). The effects of ovarian estrogens have been extensively studied in the Sex steroids encompass estrogens, androgens and progestins. These context of eating behavior. During the ovarian cycle, variations in food hormones interact with their specific receptors, namely progestin intake occur in both women and rodents. Lowest food intake is receptors (PR), androgen receptors (AR) and estrogen receptors (ER), observed following periods of high estradiol levels, namely the distributed throughout the body and the brain of both males and periovulatory phase in women and estrus in rats (Danker-Hopfe et al., females (MacLusky & McEwen, 1980; Shughrue et al., 1997; Simerly et 1995; Roney & Simmons, 2017). The anorexic effects of estradiol are al., 1990). Progestins are synthesized from cholesterol, and can be further confirmed with ovariectomy (Asarian & Geary, 2002). In rats, further converted to androgens (Miller & Auchus, 2011). Subsequently, the surgical removal of ovaries results in hyperphagia (overeating), estrogens are synthesized from androgens by the enzyme aromatase increased food reward, and body weight gain. Central administration (encoded by the CYP19A1 gene) (Simpson et al., 1994). Both males of estradiol is enough to reverse these effects, and ERs in the brain are and females produce testosterone and estrogen. In men, testes are necessary for estradiols anorexigenic effects (Palmer & Gray, 1986; producing majority of circulating androgens, while the same holds true Rivera & Eckel, 2010). The influence of androgens on eating behavior for ovaries and estrogen in premenopausal women. In contrast, in men in males have received less attention, possibly because androgen levels and postmenopausal women estrogen synthesis primarily takes place remain relatively stable throughout reproductive life and are not in other tissues than the gonads. This is possible due to the presence subject to abrupt changes as the ones associated with pregnancy or of aromatase in organs beyond the gonads, including the adipose tissue menopause (Kaufman & Vermeulen, 2005). Hypogonadism in men is and brain (Callard et al., 1978; McTernan et al., 2000; Roselli et al., associated with obesity, but castration leads to weight loss which is 1998). partially reversed with testosterone administration (Fernandez et al., 2019; Gentry & Wade, 1976; Kim et al., 2021). Notably, the Women, as well as female rodents, have fluctuating plasma levels of interpretations of testosterones effects can be complicated by sex hormones produced by the ovaries. The human menstrual cycle 3 4 Ivana Maric Ivana Maric aromatase converting it into estrogen, that holds the same anorexic factor released from the adipose tissue is leptin. Plasma leptin effect in males as it does in females. correlates to fat mass, and acts in the brain to promote catabolic effects by reducing appetite and increasing energy expenditure when fat stores Aromatase, the sole enzyme responsible for estrogen production, is are sufficient (Davis et al., 2011; Hayes et al., 2010; Rosenbaum et al., present in the brains of both women and men (Biegon, 2016). This 1996). This ideally creates a homeostatic mechanism where rising fat signifies that estrogen's actions in the brain are not solely reliant on mass leads to elevated leptin levels, subsequently curbing further peripheral sources but also involve local production. The specific weight gain. Generally, individuals suffering from obesity have functions of locally produced brain estrogen are still a subject of elevated levels of leptin due to increased fat mass, but their brains ongoing research. Interestingly, human imaging studies have hinted become resistant to its anorexic signals (Considine et al., 1996; that aromatase activity in various brain regions could be associated Matheny et al., 2011; Seeley et al., 1996). Hence, leptin therapies have with vital factors ranging from personality traits to body weight not yielded success in promoting weight loss in cases of obesity. Leptin (Biegon et al., 2020; Takahashi et al., 2018). Surprisingly, despite these levels are greatly influenced by sex hormones, with opposite response intriguing leads, there has been an absence of studies investigating the to estradiol and testosterone. Females are more sensitive to the impact of local estrogen synthesis in the brain on appetite and energy anorexic effects of leptin and central estradiol administration improves balance. Aromatase inhibitors, a commonly used class of drugs for leptin sensitivity in ovariectomized females and males (Clegg et al., breast cancer treatment, can penetrate the brain, but we know little 2006). about potential long term side effects of their action in the brain (Curtaz et al., 2022). Hence, studying the significance of local brain The gastrointestinal tract releases peptides to communicate ongoing estrogen synthesis is essential not only for a fundamental information about meals. When food reaches the duodenum, understanding of physiology but also for gaining insights into potential cholecystokinin (CCK) is immediately released to signal satiety and side effects associated with this drug treatment. inhibit eating (Kissileff et al., 1981). Close by, in the jejunum, production of glucoregulatory glucagon-like peptide 1 (GLP-1) and Peripheral signals in energy balance regulation peptide YY (PYY) takes place during and after meals to signal satiety (Woods et al., 2004). Moreover, the pancreas produces the hormones Energy balance is regulated by a complex neuroendocrine network that insulin, glucagon, somatostatin and amylin, which are not reviewed in connects peripheral organs with the central nervous system (CNS). A this thesis. continuous communication between the gastrointestinal tract, brain, and adipose tissue affects appetite, food intake and energy expenditure. Altogether, these signaling mechanisms aid the CNS in generating This regulation involves various circulating hormones that can be appropriate responses for acute meal control, and long-term control of classified as anabolic or catabolic. energy intake and expenditure to preserve homeostasis. Adipose tissue is an endocrine organ that signals to the brain, relaying information about available energy stores. A major long-term feedback 5 6 Ivana Maric Ivana Maric aromatase converting it into estrogen, that holds the same anorexic factor released from the adipose tissue is leptin. Plasma leptin effect in males as it does in females. correlates to fat mass, and acts in the brain to promote catabolic effects by reducing appetite and increasing energy expenditure when fat stores Aromatase, the sole enzyme responsible for estrogen production, is are sufficient (Davis et al., 2011; Hayes et al., 2010; Rosenbaum et al., present in the brains of both women and men (Biegon, 2016). This 1996). This ideally creates a homeostatic mechanism where rising fat signifies that estrogen's actions in the brain are not solely reliant on mass leads to elevated leptin levels, subsequently curbing further peripheral sources but also involve local production. The specific weight gain. Generally, individuals suffering from obesity have functions of locally produced brain estrogen are still a subject of elevated levels of leptin due to increased fat mass, but their brains ongoing research. Interestingly, human imaging studies have hinted become resistant to its anorexic signals (Considine et al., 1996; that aromatase activity in various brain regions could be associated Matheny et al., 2011; Seeley et al., 1996). Hence, leptin therapies have with vital factors ranging from personality traits to body weight not yielded success in promoting weight loss in cases of obesity. Leptin (Biegon et al., 2020; Takahashi et al., 2018). Surprisingly, despite these levels are greatly influenced by sex hormones, with opposite response intriguing leads, there has been an absence of studies investigating the to estradiol and testosterone. Females are more sensitive to the impact of local estrogen synthesis in the brain on appetite and energy anorexic effects of leptin and central estradiol administration improves balance. Aromatase inhibitors, a commonly used class of drugs for leptin sensitivity in ovariectomized females and males (Clegg et al., breast cancer treatment, can penetrate the brain, but we know little 2006). about potential long term side effects of their action in the brain (Curtaz et al., 2022). Hence, studying the significance of local brain The gastrointestinal tract releases peptides to communicate ongoing estrogen synthesis is essential not only for a fundamental information about meals. When food reaches the duodenum, understanding of physiology but also for gaining insights into potential cholecystokinin (CCK) is immediately released to signal satiety and side effects associated with this drug treatment. inhibit eating (Kissileff et al., 1981). Close by, in the jejunum, production of glucoregulatory glucagon-like peptide 1 (GLP-1) and Peripheral signals in energy balance regulation peptide YY (PYY) takes place during and after meals to signal satiety (Woods et al., 2004). Moreover, the pancreas produces the hormones Energy balance is regulated by a complex neuroendocrine network that insulin, glucagon, somatostatin and amylin, which are not reviewed in connects peripheral organs with the central nervous system (CNS). A this thesis. continuous communication between the gastrointestinal tract, brain, and adipose tissue affects appetite, food intake and energy expenditure. Altogether, these signaling mechanisms aid the CNS in generating This regulation involves various circulating hormones that can be appropriate responses for acute meal control, and long-term control of classified as anabolic or catabolic. energy intake and expenditure to preserve homeostasis. Adipose tissue is an endocrine organ that signals to the brain, relaying information about available energy stores. A major long-term feedback 5 6 Ivana Maric Ivana Maric Ghrelin hypothalamic-pituitary-adrenal (HPA) stress axis (Cabral et al., 2015; Cabral et al., 2016). Previous reports have indicated some Ghrelin, often called the hunger hormone, is the only gut peptide that inconsistency in the effects of ghrelin on anxiety. Rodent studies have promotes appetite by signaling energy deficiency and modulating food shown that ghrelin can have both anxiety-inducing (anxiogenic) and reward (Kojima et al., 1999). Intriguingly, ghrelin's influence extends anxiety-reducing (anxiolytic) effects, and these effects appear to beyond energy regulation, also encompassing mood and cognition depend on various factors, including prior exposure to stress (Carlini (Chuang & Zigman, 2010). Ghrelin is primarily synthetized by the et al., 2004; Currie et al., 2012; Lutter et al., 2008; Patterson et al., 2013). empty stomach in response to fasting, and reaches the brain where it Notably, our research group has demonstrated that ghrelin’s ability to acts on the growth hormone secretagogue receptor (ghrelin receptor, reduce anxiety-like behavior relies on the absence of food prior to GHSR) (Cummings et al., 2001). The ghrelin receptor can be found in anxiety testing (Alvarez-Crespo et al., 2012). brain regions important for energy homeostasis, reward processing, and emotionality (Zigman et al., 2006). Exogenous ghrelin Similarly to the leptin resistance that develops in obesity, there are administration into discrete brain sites expressing GHSR, for instance indications of diet-induced obesity leading to ghrelin resistance in the the arcuate nucleus of the hypothalamus (Arc), lateral hypothalamus hypothalamus of rodents (Lockie et al., 2015). Individuals with obesity (LH) ventral tegmental area (VTA), nucleus tractus solitarus (NTS), exhibit reduced circulating ghrelin at baseline, and do not undergo the hippocampus and amygdala, potently promotes appetitive behavior typical post-meal drop in plasma levels (Cummings, 2006). Some (Abizaid & Horvath, 2012; Alvarez-Crespo et al., 2012; Faulconbridge human studies have reported that following fasting, women have et al., 2003; Le May et al., 2019; López-Ferreras et al., 2017). Lack of higher ghrelin levels than men (Barkan et al., 2003; Espelund et al., ghrelin signaling disrupts the rewarding properties of natural rewards 2005). Moreover, our group has shown that the ghrelin axis is sexually such as food and mating, but also artificial rewards such as nicotine, dimorphic in rats, such that females exhibit higher plasma ghrelin levels cocaine and alcohol (Jerlhag et al., 2010; Jerlhag et al., 2009; Jerlhag & and lower hepatic expression of LEAP2 (Börchers et al., 2022). Engel, 2011). Liver-enriched antimicrobial peptide-2 (LEAP2) was recently identified as an endogenous peptide that acts as a competitive Interleukin-6 antagonist and inverse agonist of GHSR (Ge et al., 2018; M'Kadmi et al., 2019). LEAP2 works by blocking the effects of ghrelin binding, as Cytokines, including interleukin-6 (IL-6), are released from immune well as the constitutive activity of the receptor, essentially opposing its cells and are predominantly associated with inflammatory mechanisms. hunger-stimulating actions. Emerging reports have demonstrated that IL-6 plays a critical role in immune system modulation and the endogenous ghrelin access into the brain is limited (Perello et al., 2019). promotion of inflammatory reactions. However, it has also been The Arc, and specifically orexigenic neurons synthesizing agouti-gene- implicated in obesity and the control of energy balance (Benrick et al., related protein and neuropeptide Y (NPY/AgRP) (Chen et al., 2004), 2009; V. Wallenius et al., 2002). Adipose tissue serves as a significant are crucial mediators for the effects of peripheral ghrelin. Moreover, origin of IL-6, and, like circulating leptin levels, the secretion of IL-6 higher levels of peripheral ghrelin are hypothesized to access the is positively correlated with fat mass (Khaodhiar et al., 2004; paraventricular nucleus (PVH) through CSF, and modulate the Mohamed-Ali et al., 1997). 7 8 Ivana Maric Ivana Maric Ghrelin hypothalamic-pituitary-adrenal (HPA) stress axis (Cabral et al., 2015; Cabral et al., 2016). Previous reports have indicated some Ghrelin, often called the hunger hormone, is the only gut peptide that inconsistency in the effects of ghrelin on anxiety. Rodent studies have promotes appetite by signaling energy deficiency and modulating food shown that ghrelin can have both anxiety-inducing (anxiogenic) and reward (Kojima et al., 1999). Intriguingly, ghrelin's influence extends anxiety-reducing (anxiolytic) effects, and these effects appear to beyond energy regulation, also encompassing mood and cognition depend on various factors, including prior exposure to stress (Carlini (Chuang & Zigman, 2010). Ghrelin is primarily synthetized by the et al., 2004; Currie et al., 2012; Lutter et al., 2008; Patterson et al., 2013). empty stomach in response to fasting, and reaches the brain where it Notably, our research group has demonstrated that ghrelin’s ability to acts on the growth hormone secretagogue receptor (ghrelin receptor, reduce anxiety-like behavior relies on the absence of food prior to GHSR) (Cummings et al., 2001). The ghrelin receptor can be found in anxiety testing (Alvarez-Crespo et al., 2012). brain regions important for energy homeostasis, reward processing, and emotionality (Zigman et al., 2006). Exogenous ghrelin Similarly to the leptin resistance that develops in obesity, there are administration into discrete brain sites expressing GHSR, for instance indications of diet-induced obesity leading to ghrelin resistance in the the arcuate nucleus of the hypothalamus (Arc), lateral hypothalamus hypothalamus of rodents (Lockie et al., 2015). Individuals with obesity (LH) ventral tegmental area (VTA), nucleus tractus solitarus (NTS), exhibit reduced circulating ghrelin at baseline, and do not undergo the hippocampus and amygdala, potently promotes appetitive behavior typical post-meal drop in plasma levels (Cummings, 2006). Some (Abizaid & Horvath, 2012; Alvarez-Crespo et al., 2012; Faulconbridge human studies have reported that following fasting, women have et al., 2003; Le May et al., 2019; López-Ferreras et al., 2017). Lack of higher ghrelin levels than men (Barkan et al., 2003; Espelund et al., ghrelin signaling disrupts the rewarding properties of natural rewards 2005). Moreover, our group has shown that the ghrelin axis is sexually such as food and mating, but also artificial rewards such as nicotine, dimorphic in rats, such that females exhibit higher plasma ghrelin levels cocaine and alcohol (Jerlhag et al., 2010; Jerlhag et al., 2009; Jerlhag & and lower hepatic expression of LEAP2 (Börchers et al., 2022). Engel, 2011). Liver-enriched antimicrobial peptide-2 (LEAP2) was recently identified as an endogenous peptide that acts as a competitive Interleukin-6 antagonist and inverse agonist of GHSR (Ge et al., 2018; M'Kadmi et al., 2019). LEAP2 works by blocking the effects of ghrelin binding, as Cytokines, including interleukin-6 (IL-6), are released from immune well as the constitutive activity of the receptor, essentially opposing its cells and are predominantly associated with inflammatory mechanisms. hunger-stimulating actions. Emerging reports have demonstrated that IL-6 plays a critical role in immune system modulation and the endogenous ghrelin access into the brain is limited (Perello et al., 2019). promotion of inflammatory reactions. However, it has also been The Arc, and specifically orexigenic neurons synthesizing agouti-gene- implicated in obesity and the control of energy balance (Benrick et al., related protein and neuropeptide Y (NPY/AgRP) (Chen et al., 2004), 2009; V. Wallenius et al., 2002). Adipose tissue serves as a significant are crucial mediators for the effects of peripheral ghrelin. Moreover, origin of IL-6, and, like circulating leptin levels, the secretion of IL-6 higher levels of peripheral ghrelin are hypothesized to access the is positively correlated with fat mass (Khaodhiar et al., 2004; paraventricular nucleus (PVH) through CSF, and modulate the Mohamed-Ali et al., 1997). 7 8 Ivana Maric Ivana Maric Chronic low-grade inflammation associated with excessive adipose The hypothalamus tissue is a key driver of metabolic syndrome (Monteiro & Azevedo, 2010). Interestingly, central injection of IL-6 increases energy The hypothalamus was early recognized to be a crucial center for expenditure and decreases fat mass (Timper et al., 2017; K. Wallenius energy homeostasis. Lesioning studies of the ventromedial et al., 2002). It's possible that the primary source of centrally acting IL- hypothalamic area (VMH) demonstrated animals gaining weight due to 6 comes from local production in the brain. This is suggested by the hyperphagia and reduced energy expenditure, whereas lesions of the observation that IL-6 levels in the cerebrospinal fluid (CSF) are higher LH produced the opposite effects (Anand & Brobeck, 1951; Cox & than the concentrations found peripherally. Indeed, brain regions Sims, 1988; Hetherington & Ranson, 1940). Receptors for the crucial for energy regulation, like the LH, produce this cytokine adiposity signal, leptin, were found to be most abundant in the Arc (López-Ferreras et al., 2021; Miyahara et al., 2000). In contrast to the (Elmquist et al., 1999). Furthermore, two important neuronal positive correlation of obesity and serum IL-6, the opposite stands true populations, sensitive to leptin and other peripheral signals such as for IL-6 levels in the CSF of obese men (Stenlöf et al., 2003). It is worth ghrelin, were identified in the Arc: anorexigenic POMC/CART noting that the limited research on central IL-6 and its role in obesity neurons and orexigenic NPY/AgRP neurons (Mercer et al., 1996; has almost exclusively concentrated on male subjects. Seeley et al., 1997). Supported by the initial lesion studies of the LH, which resulted in weight loss, it is evident that the LH primarily Central nervous system integration functions as an anabolic region that also drives reward (Margules & Olds, 1962). Within the LH the orexigenic peptides melanin- Even though we know better, we often eat too much. Food intake is concentrating hormone (MCH) and orexins (ORX) are expressed (Qu driven not only by the necessity to restore energy balance but also by et al., 1996; Toshinai et al., 2003). On the other hand, neurons in the the allure of palatable foods, which can continue to stimulate PVH express primarily anorexigenic neuroactive substances, including consumption even when a sense of fullness is present. Indeed, in the corticotropin-releasing hormone (CRH) and oxytocin (OT) (Hill, case of obesity, negative feedback from metabolic satiety signals like 2012). The hypothalamic brain regions are strongly interconnected, leptin proves ineffective in maintaining energy balance. Feeding and receive and send projections among one another but also to the behavior control is orchestrated by an extensive neural network that forebrain and hindbrain. spans from basal forebrain to the caudal brainstem. The scientific literature often refers to two types of behavioral control, that are The hindbrain closely intertwined: homeostatic mechanisms that responds to variations in metabolic need, and hedonic mechanisms that rely on The brainstem receives sensory information from the periphery and cognition and the pleasure derived from food. coordinates autonomic functions such as heart rate, digestion, and metabolism. It is shown to serve a crucial role in the regulation of meal size and thermogenesis. Research conducted by Grill and colleagues using the chronic decerebrate (CD) rat model, where the caudal brainstem is surgically isolated from the forebrain, highlighted the 9 10 Ivana Maric Ivana Maric Chronic low-grade inflammation associated with excessive adipose The hypothalamus tissue is a key driver of metabolic syndrome (Monteiro & Azevedo, 2010). Interestingly, central injection of IL-6 increases energy The hypothalamus was early recognized to be a crucial center for expenditure and decreases fat mass (Timper et al., 2017; K. Wallenius energy homeostasis. Lesioning studies of the ventromedial et al., 2002). It's possible that the primary source of centrally acting IL- hypothalamic area (VMH) demonstrated animals gaining weight due to 6 comes from local production in the brain. This is suggested by the hyperphagia and reduced energy expenditure, whereas lesions of the observation that IL-6 levels in the cerebrospinal fluid (CSF) are higher LH produced the opposite effects (Anand & Brobeck, 1951; Cox & than the concentrations found peripherally. Indeed, brain regions Sims, 1988; Hetherington & Ranson, 1940). Receptors for the crucial for energy regulation, like the LH, produce this cytokine adiposity signal, leptin, were found to be most abundant in the Arc (López-Ferreras et al., 2021; Miyahara et al., 2000). In contrast to the (Elmquist et al., 1999). Furthermore, two important neuronal positive correlation of obesity and serum IL-6, the opposite stands true populations, sensitive to leptin and other peripheral signals such as for IL-6 levels in the CSF of obese men (Stenlöf et al., 2003). It is worth ghrelin, were identified in the Arc: anorexigenic POMC/CART noting that the limited research on central IL-6 and its role in obesity neurons and orexigenic NPY/AgRP neurons (Mercer et al., 1996; has almost exclusively concentrated on male subjects. Seeley et al., 1997). Supported by the initial lesion studies of the LH, which resulted in weight loss, it is evident that the LH primarily Central nervous system integration functions as an anabolic region that also drives reward (Margules & Olds, 1962). Within the LH the orexigenic peptides melanin- Even though we know better, we often eat too much. Food intake is concentrating hormone (MCH) and orexins (ORX) are expressed (Qu driven not only by the necessity to restore energy balance but also by et al., 1996; Toshinai et al., 2003). On the other hand, neurons in the the allure of palatable foods, which can continue to stimulate PVH express primarily anorexigenic neuroactive substances, including consumption even when a sense of fullness is present. Indeed, in the corticotropin-releasing hormone (CRH) and oxytocin (OT) (Hill, case of obesity, negative feedback from metabolic satiety signals like 2012). The hypothalamic brain regions are strongly interconnected, leptin proves ineffective in maintaining energy balance. Feeding and receive and send projections among one another but also to the behavior control is orchestrated by an extensive neural network that forebrain and hindbrain. spans from basal forebrain to the caudal brainstem. The scientific literature often refers to two types of behavioral control, that are The hindbrain closely intertwined: homeostatic mechanisms that responds to variations in metabolic need, and hedonic mechanisms that rely on The brainstem receives sensory information from the periphery and cognition and the pleasure derived from food. coordinates autonomic functions such as heart rate, digestion, and metabolism. It is shown to serve a crucial role in the regulation of meal size and thermogenesis. Research conducted by Grill and colleagues using the chronic decerebrate (CD) rat model, where the caudal brainstem is surgically isolated from the forebrain, highlighted the 9 10 Ivana Maric Ivana Maric significant role of the brainstem in regulating feeding behavior (Grill, LOCUS COERULEUS 1980; Kaplan et al., 1993). The CD rat does not initiate meals on its Adjacent with PBN is the major source of noradrenergic innervation own and does not engage in compensatory food intake following in the brain, the locus coeruleus (LC). It is crucial for arousal, and thus fasting. However, the CD rats were able to adjust meal size according implicated in pathologies related with hyperarousal such as anxiety to energy density and reduce intake following leptin administration. disorders (Morris et al., 2020). Interestingly, anxiety disorders are more Taken together, this suggests that the caudal brainstem is sufficient for prevalent in women, and the LC is well-investigated in regards to its the sensory feedback related to taste and ingestion, as well as some sexual dimorphism (Bangasser et al., 2016). Stress alters LC activity, an aspects of nutrient sensing. However, without communication with the effect mediated by CRF, and activates downstream targets to cause forebrain there is a lack of adaptive responses to changing energy vigilance (Koob, 1999). Studies have shown that the female LC needs. The NTS is the brainstem region most thoroughly investigated displays increased sensitivity to the effects of CRF, and this heightened in relation to eating behavior. It is the afferent center of the vagus sensitivity is not associated with activational hormonal levels but rather nerve, and receives direct sensory inputs related to food intake, such with organizational differences (Bangasser et al., 2010; Bangasser et al., as stomach distention (Huo et al., 2007). Furthermore, the NTS 2013; Curtis et al., 2006). Recently, the LC has started receiving integrates various signals from the periphery, it reacts to changes in attention in the field of feeding. It was reported that LC activity is blood glucose levels and peripheral feeding peptides (Rinaman, 2010). necessary for fear-induced feeding suppression through projections to In addition to being produced in the intestines, the anorexic hormone PBN (Yang et al., 2021). Moreover, Sciolino and colleagues GLP-1 is synthesized within neurons in the NTS. This central demonstrated that satiety can modulate LC activity, and identified that production of GLP-1 is believed to be the primary source acting in the activation of noradrenergic projections to the LH suppressed feeding brain, as GLP-1 released by the intestines is likely metabolized before and triggered anxiety-like behavior (Sciolino et al., 2022). it can reach the brain (Holt et al., 2019). The reward system PARABRACHIAL NUCLEUS The parabrachial nucleus (PBN) is an important hub for integrating The mesolimbic system, often referred to as the reward system, interoceptive and exteroceptive inputs. It responds to taste, thermal comprises neural networks that play a key role in regulating the sensation, visceral malaise, arousal and energy status. Moreover, PBN physiological and cognitive aspects of reward processing. These neural receives meal-related satiety signals from the NTS and integrates substrates are activated both prior to and following the receipt of a them with various neurochemical signals, including leptin (Alhadeff reward, establishing connections between stimuli and pleasure, et al., 2014). One important aspect of feeding control imposed by ultimately prompting behaviors that fuel the pursuit of the rewarding PBN is aversion-induced anorexia, such that neurons in this area stimuli in question. Eating behavior, like drinking, sleeping and mating, terminate feeding in response to pain, heat and stress (Chen et al., represents natural reinforcements essential for the preservation of a 2018; Jen-Hui et al., 2023). species. Artificial rewards, like substances of abuse, have the potential to hijack the reward system that originally evolved to regulate motivation for the natural rewards necessary for survival (Koob, 1992). 11 12 Ivana Maric Ivana Maric significant role of the brainstem in regulating feeding behavior (Grill, LOCUS COERULEUS 1980; Kaplan et al., 1993). The CD rat does not initiate meals on its Adjacent with PBN is the major source of noradrenergic innervation own and does not engage in compensatory food intake following in the brain, the locus coeruleus (LC). It is crucial for arousal, and thus fasting. However, the CD rats were able to adjust meal size according implicated in pathologies related with hyperarousal such as anxiety to energy density and reduce intake following leptin administration. disorders (Morris et al., 2020). Interestingly, anxiety disorders are more Taken together, this suggests that the caudal brainstem is sufficient for prevalent in women, and the LC is well-investigated in regards to its the sensory feedback related to taste and ingestion, as well as some sexual dimorphism (Bangasser et al., 2016). Stress alters LC activity, an aspects of nutrient sensing. However, without communication with the effect mediated by CRF, and activates downstream targets to cause forebrain there is a lack of adaptive responses to changing energy vigilance (Koob, 1999). Studies have shown that the female LC needs. The NTS is the brainstem region most thoroughly investigated displays increased sensitivity to the effects of CRF, and this heightened in relation to eating behavior. It is the afferent center of the vagus sensitivity is not associated with activational hormonal levels but rather nerve, and receives direct sensory inputs related to food intake, such with organizational differences (Bangasser et al., 2010; Bangasser et al., as stomach distention (Huo et al., 2007). Furthermore, the NTS 2013; Curtis et al., 2006). Recently, the LC has started receiving integrates various signals from the periphery, it reacts to changes in attention in the field of feeding. It was reported that LC activity is blood glucose levels and peripheral feeding peptides (Rinaman, 2010). necessary for fear-induced feeding suppression through projections to In addition to being produced in the intestines, the anorexic hormone PBN (Yang et al., 2021). Moreover, Sciolino and colleagues GLP-1 is synthesized within neurons in the NTS. This central demonstrated that satiety can modulate LC activity, and identified that production of GLP-1 is believed to be the primary source acting in the activation of noradrenergic projections to the LH suppressed feeding brain, as GLP-1 released by the intestines is likely metabolized before and triggered anxiety-like behavior (Sciolino et al., 2022). it can reach the brain (Holt et al., 2019). The reward system PARABRACHIAL NUCLEUS The parabrachial nucleus (PBN) is an important hub for integrating The mesolimbic system, often referred to as the reward system, interoceptive and exteroceptive inputs. It responds to taste, thermal comprises neural networks that play a key role in regulating the sensation, visceral malaise, arousal and energy status. Moreover, PBN physiological and cognitive aspects of reward processing. These neural receives meal-related satiety signals from the NTS and integrates substrates are activated both prior to and following the receipt of a them with various neurochemical signals, including leptin (Alhadeff reward, establishing connections between stimuli and pleasure, et al., 2014). One important aspect of feeding control imposed by ultimately prompting behaviors that fuel the pursuit of the rewarding PBN is aversion-induced anorexia, such that neurons in this area stimuli in question. Eating behavior, like drinking, sleeping and mating, terminate feeding in response to pain, heat and stress (Chen et al., represents natural reinforcements essential for the preservation of a 2018; Jen-Hui et al., 2023). species. Artificial rewards, like substances of abuse, have the potential to hijack the reward system that originally evolved to regulate motivation for the natural rewards necessary for survival (Koob, 1992). 11 12 Ivana Maric Ivana Maric The mesolimbic dopamine pathway is centered around the VTA (Wise, The sexual dimorphism of adiposity 2004). Dopaminergic neurons originating from the VTA project to brain regions associated with executive, emotional, and motivational Appetite, once essential for preventing starvation, can become functions (Wise & Bozarth, 1984). Projections from the VTA to the maladaptive in our modern, food-abundant society and result in nucleus accumbens (NAc) are well recognized for goal-directed overeating and, ultimately, obesity. The incidence of overweight and motivated behaviors (Bardo et al., 1996). Moreover, bidirectional obesity has increased dramatically in recent years, and the World communication takes place between the VTA and the amygdala, Health Organization (WHO) estimates that approximately 1.9 billion hippocampus, prefrontal cortex (PFC), and hypothalamus (Alonso- adults worldwide are overweight. This escalating concern is not to be Alonso et al., 2015). Hence, the regulation of food intake and energy taken lightly, given the strong correlations between obesity and expenditure is under the combined control of energy status, emotional cardiovascular diseases, diabetes, some cancers, and autoimmune and processes, memory, cognition, and reward responses. neurodegenerative disorders. Moreover, epidemiological studies indicate a bidirectional relationship between excessive body weight and Metabolic signals involved in the homeostatic regulation of energy psychiatric disorders, alluding to the presence of shared mechanisms balance discussed earlier, are also implicated in the rewarding aspect of underpinning these seemingly distinct conditions (Milaneschi et al., eating. Leptin, insulin, ghrelin, and GLP-1 all possess receptors within 2019). While there is a myriad of potential contributors to obesity, the the mesolimbic pathway and act there to modulate food seeking and heightened availability and consumption of food is frequently motivation to work for food (Alhadeff et al., 2012; Dossat et al., 2013; identified as a prime suspect for the current epidemic. In fact, studies Fulton et al., 2006; Palmiter, 2007; Skibicka et al., 2011). Remarkably, show that consumption of a high-fat high-sugar diet can lead to GLP-1R and GHSR have been found to exert influence beyond metabolic consequences independent of obesity development, regulating food rewards and have gained attention in their ability to suggesting that unhealthy food per se can have adverse effects on modulate the effects of addictive substances as well (Davis et al., 2007; physiology (la Fleur et al., 2011). Nonetheless, due to its palatability, Graham et al., 2013; Jerlhag et al., 2010; Klausen et al., 2022). the Western diet promotes overeating in susceptible individuals and results in a state of positive energy balance and adiposity, despite the Previous reports, suggest that females may have a more hedonically- many feedback mechanisms to maintain homeostasis discussed above. driven feeding behavior (Buczek et al., 2020). For instance female rats show higher activation of reward-related brain regions and a stronger Excess energy is primarily stored as triglycerides in white adipose tissue shift for palatable food in a conditioned place preference (Sinclair et (WAT), but the location of this fat storage matters significantly for al., 2017). Ovarian estradiol modulates reward derived from food, and health outcomes. Visceral fat, which accumulates in the abdominal estradiol administration selectively targeting the VTA of female rats region, is strongly linked to metabolic disorders, while subcutaneous reduces operant behavior for sucrose in the progressive ratio (Richard fat, found just beneath the skin, appears to offer some protection et al., 2017). against these conditions (Frank et al., 2019). Interestingly, there is a clear sexual dimorphism in fat distribution (Palmer & Clegg, 2015). Men, despite generally having lower overall body fat, are prone to 13 14 Ivana Maric Ivana Maric The mesolimbic dopamine pathway is centered around the VTA (Wise, The sexual dimorphism of adiposity 2004). Dopaminergic neurons originating from the VTA project to brain regions associated with executive, emotional, and motivational Appetite, once essential for preventing starvation, can become functions (Wise & Bozarth, 1984). Projections from the VTA to the maladaptive in our modern, food-abundant society and result in nucleus accumbens (NAc) are well recognized for goal-directed overeating and, ultimately, obesity. The incidence of overweight and motivated behaviors (Bardo et al., 1996). Moreover, bidirectional obesity has increased dramatically in recent years, and the World communication takes place between the VTA and the amygdala, Health Organization (WHO) estimates that approximately 1.9 billion hippocampus, prefrontal cortex (PFC), and hypothalamus (Alonso- adults worldwide are overweight. This escalating concern is not to be Alonso et al., 2015). Hence, the regulation of food intake and energy taken lightly, given the strong correlations between obesity and expenditure is under the combined control of energy status, emotional cardiovascular diseases, diabetes, some cancers, and autoimmune and processes, memory, cognition, and reward responses. neurodegenerative disorders. Moreover, epidemiological studies indicate a bidirectional relationship between excessive body weight and Metabolic signals involved in the homeostatic regulation of energy psychiatric disorders, alluding to the presence of shared mechanisms balance discussed earlier, are also implicated in the rewarding aspect of underpinning these seemingly distinct conditions (Milaneschi et al., eating. Leptin, insulin, ghrelin, and GLP-1 all possess receptors within 2019). While there is a myriad of potential contributors to obesity, the the mesolimbic pathway and act there to modulate food seeking and heightened availability and consumption of food is frequently motivation to work for food (Alhadeff et al., 2012; Dossat et al., 2013; identified as a prime suspect for the current epidemic. In fact, studies Fulton et al., 2006; Palmiter, 2007; Skibicka et al., 2011). Remarkably, show that consumption of a high-fat high-sugar diet can lead to GLP-1R and GHSR have been found to exert influence beyond metabolic consequences independent of obesity development, regulating food rewards and have gained attention in their ability to suggesting that unhealthy food per se can have adverse effects on modulate the effects of addictive substances as well (Davis et al., 2007; physiology (la Fleur et al., 2011). Nonetheless, due to its palatability, Graham et al., 2013; Jerlhag et al., 2010; Klausen et al., 2022). the Western diet promotes overeating in susceptible individuals and results in a state of positive energy balance and adiposity, despite the Previous reports, suggest that females may have a more hedonically- many feedback mechanisms to maintain homeostasis discussed above. driven feeding behavior (Buczek et al., 2020). For instance female rats show higher activation of reward-related brain regions and a stronger Excess energy is primarily stored as triglycerides in white adipose tissue shift for palatable food in a conditioned place preference (Sinclair et (WAT), but the location of this fat storage matters significantly for al., 2017). Ovarian estradiol modulates reward derived from food, and health outcomes. Visceral fat, which accumulates in the abdominal estradiol administration selectively targeting the VTA of female rats region, is strongly linked to metabolic disorders, while subcutaneous reduces operant behavior for sucrose in the progressive ratio (Richard fat, found just beneath the skin, appears to offer some protection et al., 2017). against these conditions (Frank et al., 2019). Interestingly, there is a clear sexual dimorphism in fat distribution (Palmer & Clegg, 2015). Men, despite generally having lower overall body fat, are prone to 13 14 Ivana Maric Ivana Maric storing fat in the visceral depot. When storage becomes excessive, is adipocytes contain high amounts of mitochondrial uncoupling protein associated with an increased inflammatory state and endocrine release 1 (UCP-1), that following sympathetic stimulation allows for heat of pro-inflammatory metabolites, which can ultimately cause generation utilizing fatty acids and glucose (Chondronikola et al., 2016; complications such as insulin resistance (Foster & Pagliassotti, 2012). Hanssen et al., 2015). This unique property is essential to maintain On the other hand, premenopausal women tend to favor subcutaneous body temperature, and enable survival in cold environments. Notably, fat depots, which possess better adaptability for growth and, thus, a cold-exposure and the concurrent BAT activation has been proven metabolically healthier phenotype. However, with the onset of advantageous for glucose homeostasis and insulin sensitivity in both menopause, there is a shift in fat storage that aligns postmenopausal healthy individuals and individuals with type 2 diabetes women more closely with men in terms of metabolic risk. The timing (Chondronikola et al., 2014). Therefore, it is rather unsurprising that of these changes implies involvement of ovarian hormones, and the the activity and prevalence of BAT is found to be lower in older and fact that estrogen replacement therapy can mitigate these effects obese populations (Pfannenberg et al., 2010). BAT research in humans emphasizes the pivotal role of this hormone in particular (Gambacciani has experienced a revival in recent years, reigniting interest for using et al., 1997). this tissue as a potential therapeutic target for boosting energy expenditure and promoting weight loss. Intriguingly, women appear to One could speculate that the evolutionary basis to the sexually have higher BAT mass and thermogenic activity (Pfannenberg et al., dimorphic white adipose tissue deposition could be due to the 2010). Further human studies are needed to elucidate the molecular differences in lipolytic activity between the depots and the presumed basis of these differences, but rodent studies indicate that female BAT roles of our prehistorical ancestors. In men, the evolutionary forces may contain higher mitochondrial density, UCP-1 expression and is driving the accumulation of visceral fat may be rooted in its rapid more sensitive to β-adrenergic stimulation (Rodriguez-Cuenca et al., mobilization capability, useful for shorter-term energetic challenges 2002) such as perilous hunting scenarios. Conversely, women had to survive pregnancy and lactation in a very challenging environment. There is a The sex bias in biomedical research close connection between reproduction and adiposity, women suffering from anorexia nervosa stop ovulating due to a decline in Out of the ten prescription drugs pulled from the U.S. market between leptin signaling insufficient fat depots to support a pregnancy (Frisch, 1997 and 2001, eight were withdrawn due to health risks affecting 1990). On the other hand, pregnancy leads to growing subcutaneous women (U.S. Government Accountability Office, 2001). It is adipose tissue depots that can be utilized during the energy demanding astonishing that, despite the rigorous and expensive drug development period of lactation. process, we fail to detect severe side effects in half of the population until the drug is already on the market. Throughout history, women While WAT is an endocrine organ that functions as a storage reservoir have been neglected in biological research (Liu & Mager, 2016). It for excess energy, brown adipose tissue (BAT) can expend energy via wasn't until the 1990s that the inclusion of female participants in nonshivering thermogenesis during cold exposure (Cannon & federally supported phase III clinical trials became obligatory, as Nedergaard, 2004; van Marken Lichtenbelt et al., 2009). Brown mandated by the US National Institute of Health Revitalization Act of 15 16 Ivana Maric Ivana Maric storing fat in the visceral depot. When storage becomes excessive, is adipocytes contain high amounts of mitochondrial uncoupling protein associated with an increased inflammatory state and endocrine release 1 (UCP-1), that following sympathetic stimulation allows for heat of pro-inflammatory metabolites, which can ultimately cause generation utilizing fatty acids and glucose (Chondronikola et al., 2016; complications such as insulin resistance (Foster & Pagliassotti, 2012). Hanssen et al., 2015). This unique property is essential to maintain On the other hand, premenopausal women tend to favor subcutaneous body temperature, and enable survival in cold environments. Notably, fat depots, which possess better adaptability for growth and, thus, a cold-exposure and the concurrent BAT activation has been proven metabolically healthier phenotype. However, with the onset of advantageous for glucose homeostasis and insulin sensitivity in both menopause, there is a shift in fat storage that aligns postmenopausal healthy individuals and individuals with type 2 diabetes women more closely with men in terms of metabolic risk. The timing (Chondronikola et al., 2014). Therefore, it is rather unsurprising that of these changes implies involvement of ovarian hormones, and the the activity and prevalence of BAT is found to be lower in older and fact that estrogen replacement therapy can mitigate these effects obese populations (Pfannenberg et al., 2010). BAT research in humans emphasizes the pivotal role of this hormone in particular (Gambacciani has experienced a revival in recent years, reigniting interest for using et al., 1997). this tissue as a potential therapeutic target for boosting energy expenditure and promoting weight loss. Intriguingly, women appear to One could speculate that the evolutionary basis to the sexually have higher BAT mass and thermogenic activity (Pfannenberg et al., dimorphic white adipose tissue deposition could be due to the 2010). Further human studies are needed to elucidate the molecular differences in lipolytic activity between the depots and the presumed basis of these differences, but rodent studies indicate that female BAT roles of our prehistorical ancestors. In men, the evolutionary forces may contain higher mitochondrial density, UCP-1 expression and is driving the accumulation of visceral fat may be rooted in its rapid more sensitive to β-adrenergic stimulation (Rodriguez-Cuenca et al., mobilization capability, useful for shorter-term energetic challenges 2002) such as perilous hunting scenarios. Conversely, women had to survive pregnancy and lactation in a very challenging environment. There is a The sex bias in biomedical research close connection between reproduction and adiposity, women suffering from anorexia nervosa stop ovulating due to a decline in Out of the ten prescription drugs pulled from the U.S. market between leptin signaling insufficient fat depots to support a pregnancy (Frisch, 1997 and 2001, eight were withdrawn due to health risks affecting 1990). On the other hand, pregnancy leads to growing subcutaneous women (U.S. Government Accountability Office, 2001). It is adipose tissue depots that can be utilized during the energy demanding astonishing that, despite the rigorous and expensive drug development period of lactation. process, we fail to detect severe side effects in half of the population until the drug is already on the market. Throughout history, women While WAT is an endocrine organ that functions as a storage reservoir have been neglected in biological research (Liu & Mager, 2016). It for excess energy, brown adipose tissue (BAT) can expend energy via wasn't until the 1990s that the inclusion of female participants in nonshivering thermogenesis during cold exposure (Cannon & federally supported phase III clinical trials became obligatory, as Nedergaard, 2004; van Marken Lichtenbelt et al., 2009). Brown mandated by the US National Institute of Health Revitalization Act of 15 16 Ivana Maric Ivana Maric 1993. Evidence shows that excluding women from trials has led to an AIM unrepresentative assessment of drug efficacy and side effects (Correa- De-Araujo, 2006). The overarching aim of this thesis was to identify sex differences in metabolic and behavioral control within rodent models of obesity and The underrepresentation of females in basic scientific studies and anxiety. animal disease models is remarkable and remains an ongoing issue. A bibliometric analysis from 2011 reported that a selective focus on male The specific aims were: rodents was observed in a striking 80% of published neuroscience studies (Beery & Zucker, 2011). Subsequently, the US National Paper I Institutes of Health introduced a policy requiring funding recipients to To determine whether characteristics of diet-induced obesity differ consider sex as a biological variable (Clayton & Collins, 2014). between both sexes of rats and mice, and to investigate the potential Nevertheless, despite improvements in the inclusion of females, energy balance disturbances underlying the observed divergence. significant gaps persist in the reporting and analysis of data by sex (Woitowich et al., 2020). Paper II Considering that pre-clinical research marks the initial stage in the To explore if diet-induced obesity induces changes in the expression development of a new treatment, this bias creates a scenario where of interleukin-6 within specific brain regions, in both male and female drugs are developed based on male biology primarily. Without rodents. Additionally, we sought to determine whether interleukin-6 considering the role of sex, there is a risk of assuming a general effect within the parabrachial nucleus is necessary for maintaining normal when it only applies to one sex. Moreover, lack of sex analysis can lead energy balance. to mistakenly disregarding treatments when there are offsetting effects in the two sexes. An example that illustrates this issue is the pain Paper III medication MorphiDex that failed late-stage clinical trials (Galer et al., To examine if ghrelin signaling in the locus coeruleus mediates 2005; Institute of Medicine Forum on & Nervous System, 2011). ingestive, reward and anxiety-like behavior, and whether these effects Although women were enrolled, the data was not analyzed by sex. vary between male and female rats. None of animal studies conducted prior to the clinical trials had involved females (Mogil, 2020), and it was not until after the Paper IV unsuccessful clinical trials that it became evident that the effect of the To investigate the influence of local brain estrogen synthesis on eating active components could not be demonstrated in female rodents behavior in both male and female rats, with a specific focus on the role (Grisel et al., 2005). Therefore, it is quite likely that an effect in men of aromatase in the amygdala. may have been cancelled out when combined with the data of women. This is likely one of many cases where a drug with the potential to benefit one sex was disregarded in clinical trials. 17 18 Ivana Maric Ivana Maric 1993. Evidence shows that excluding women from trials has led to an AIM unrepresentative assessment of drug efficacy and side effects (Correa- De-Araujo, 2006). The overarching aim of this thesis was to identify sex differences in metabolic and behavioral control within rodent models of obesity and The underrepresentation of females in basic scientific studies and anxiety. animal disease models is remarkable and remains an ongoing issue. A bibliometric analysis from 2011 reported that a selective focus on male The specific aims were: rodents was observed in a striking 80% of published neuroscience studies (Beery & Zucker, 2011). Subsequently, the US National Paper I Institutes of Health introduced a policy requiring funding recipients to To determine whether characteristics of diet-induced obesity differ consider sex as a biological variable (Clayton & Collins, 2014). between both sexes of rats and mice, and to investigate the potential Nevertheless, despite improvements in the inclusion of females, energy balance disturbances underlying the observed divergence. significant gaps persist in the reporting and analysis of data by sex (Woitowich et al., 2020). Paper II Considering that pre-clinical research marks the initial stage in the To explore if diet-induced obesity induces changes in the expression development of a new treatment, this bias creates a scenario where of interleukin-6 within specific brain regions, in both male and female drugs are developed based on male biology primarily. Without rodents. Additionally, we sought to determine whether interleukin-6 considering the role of sex, there is a risk of assuming a general effect within the parabrachial nucleus is necessary for maintaining normal when it only applies to one sex. Moreover, lack of sex analysis can lead energy balance. to mistakenly disregarding treatments when there are offsetting effects in the two sexes. An example that illustrates this issue is the pain Paper III medication MorphiDex that failed late-stage clinical trials (Galer et al., To examine if ghrelin signaling in the locus coeruleus mediates 2005; Institute of Medicine Forum on & Nervous System, 2011). ingestive, reward and anxiety-like behavior, and whether these effects Although women were enrolled, the data was not analyzed by sex. vary between male and female rats. None of animal studies conducted prior to the clinical trials had involved females (Mogil, 2020), and it was not until after the Paper IV unsuccessful clinical trials that it became evident that the effect of the To investigate the influence of local brain estrogen synthesis on eating active components could not be demonstrated in female rodents behavior in both male and female rats, with a specific focus on the role (Grisel et al., 2005). Therefore, it is quite likely that an effect in men of aromatase in the amygdala. may have been cancelled out when combined with the data of women. This is likely one of many cases where a drug with the potential to benefit one sex was disregarded in clinical trials. 17 18 Ivana Maric Ivana Maric METHODOLOGICAL light cycle and still consume approximately 25% of their daily intake during this period (Sidlo et al., 1995). Moreover, we tried to avoid CONSIDERATIONS individual housing in experiments not requiring it, and made sure that our study design aligned with the 3R principle in mind (replace, reduce, This section provides a general overview and rationale of the methods refine), to minimize animal suffering. used in this thesis. For specific procedural details, please refer to the "Materials and Methods" sections in each respective paper. All studies conformed to and received approval by the local Ethics Committee for animal care at the Institute of Experimental Experimental model Biomedicine at the University of Gothenburg in Sweden. Additionally, a big part of the experiments in Paper III and IV were performed at The work of this thesis is based on studies carried out using male and Pennsylvania State University and received approval by the local female Sprague Dawley rats, as well as C57BL/6N mice (Paper I and Institutional Animal Care and Use Committee. Ethical permit numbers II). The rodent neural circuits and neurotransmitter systems involved are specified in each paper. in eating behavior and anxiety are similar to those in humans. For example, monogenetic mutations that induce obesity in humans do so Obesogenic diets in rodents as well (Krude et al., 1998; Montague et al., 1997; Yaswen et al., 1999). As evidenced by the findings in Paper I, variations do While animal models with single-gene mutations are useful for exist in diet-induced obesity between mice and rats, and their potential studying the specific contribution of certain genes to energy for translational relevance may differ across various disease aspects. metabolism, they fall short in mimicking the complexity of the current There are already established differences between mice and rats in obesity epidemic. Instead, the diet-induced obesity paradigm provides terms of ingestive behavior, for instance mice tend to eat smaller, more a model that better replicates interactions between polygenetic frequent, meals than rats. Hence, to enhance confidence in the predispositions and weight gain in an obesogenic environment. In translatability of novel findings and drug effects to humans, it is laboratory settings, a variety of palatable diets are utilized to induce advisable to confirm them in both species. Furthermore, certain obesity in rodents, all sharing common characteristics of being high in practical considerations in the experimental setup may raise questions energy content and abundant in sugars and fats. "The 'cafeteria diet,' about translational relevance. For instance, the need to individually first introduced by Sclafani and Springer in 1976, comprises an animals after some surgeries (Paper II, III and IV), which affects their unstandardized combination of unhealthy food items people emotional state, is notable given the social nature of rats. Additionally, commonly consume, such as cookies, candy, and chips (Sclafani & conducting all experiments during the light cycle, despite rodents being Springer, 1976). Nowadays, commercially produced energy-dense nocturnal animals, might not mimic natural conditions very well. pellets are used more regularly. The most common pellets contain Although rodents are more active during the dark cycle and typically either 45% or 60% fat by energy, and are therefore classified as 'high- consume the majority of their daily food intake then, it is important to fat diets'. Nevertheless, it is worth noting that while their carbohydrate note that rats do not have consolidated sleep patterns throughout the content (20%) is lower than their fat content, these diets do contain 19 20 Ivana Maric Ivana Maric METHODOLOGICAL light cycle and still consume approximately 25% of their daily intake during this period (Sidlo et al., 1995). Moreover, we tried to avoid CONSIDERATIONS individual housing in experiments not requiring it, and made sure that our study design aligned with the 3R principle in mind (replace, reduce, This section provides a general overview and rationale of the methods refine), to minimize animal suffering. used in this thesis. For specific procedural details, please refer to the "Materials and Methods" sections in each respective paper. All studies conformed to and received approval by the local Ethics Committee for animal care at the Institute of Experimental Experimental model Biomedicine at the University of Gothenburg in Sweden. Additionally, a big part of the experiments in Paper III and IV were performed at The work of this thesis is based on studies carried out using male and Pennsylvania State University and received approval by the local female Sprague Dawley rats, as well as C57BL/6N mice (Paper I and Institutional Animal Care and Use Committee. Ethical permit numbers II). The rodent neural circuits and neurotransmitter systems involved are specified in each paper. in eating behavior and anxiety are similar to those in humans. For example, monogenetic mutations that induce obesity in humans do so Obesogenic diets in rodents as well (Krude et al., 1998; Montague et al., 1997; Yaswen et al., 1999). As evidenced by the findings in Paper I, variations do While animal models with single-gene mutations are useful for exist in diet-induced obesity between mice and rats, and their potential studying the specific contribution of certain genes to energy for translational relevance may differ across various disease aspects. metabolism, they fall short in mimicking the complexity of the current There are already established differences between mice and rats in obesity epidemic. Instead, the diet-induced obesity paradigm provides terms of ingestive behavior, for instance mice tend to eat smaller, more a model that better replicates interactions between polygenetic frequent, meals than rats. Hence, to enhance confidence in the predispositions and weight gain in an obesogenic environment. In translatability of novel findings and drug effects to humans, it is laboratory settings, a variety of palatable diets are utilized to induce advisable to confirm them in both species. Furthermore, certain obesity in rodents, all sharing common characteristics of being high in practical considerations in the experimental setup may raise questions energy content and abundant in sugars and fats. "The 'cafeteria diet,' about translational relevance. For instance, the need to individually first introduced by Sclafani and Springer in 1976, comprises an animals after some surgeries (Paper II, III and IV), which affects their unstandardized combination of unhealthy food items people emotional state, is notable given the social nature of rats. Additionally, commonly consume, such as cookies, candy, and chips (Sclafani & conducting all experiments during the light cycle, despite rodents being Springer, 1976). Nowadays, commercially produced energy-dense nocturnal animals, might not mimic natural conditions very well. pellets are used more regularly. The most common pellets contain Although rodents are more active during the dark cycle and typically either 45% or 60% fat by energy, and are therefore classified as 'high- consume the majority of their daily food intake then, it is important to fat diets'. Nevertheless, it is worth noting that while their carbohydrate note that rats do not have consolidated sleep patterns throughout the content (20%) is lower than their fat content, these diets do contain 19 20 Ivana Maric Ivana Maric simple sugars (sucrose) as opposed to regular chow pellets, making All surgeries were performed under ketamine/xylazine anesthesia them more than just high-fat diets. Another vital element in inducing administered intraperitonially. It is common practice in our lab to overeating and emulating the obesogenic conditions in humans is the administer a dosage that is up to 15% lower, per kilogram, to female emphasis on variety, often referred to as the 'buffet effect'. With this rats. However, there is no published data that confirms our observed concept in mind, Susanne la Fleur's laboratory introduced a free-choice sex difference in anesthesia sensitivity. A rat brain atlas (Paxinos & high-fat high-sugar diet, offering lard and a 30% sucrose solution with Watson) was used for decision of appropriate coordinates to target the standard chow. This approach demonstrated a more sustained lateral parabrachial nucleus and paraventricular nucleus (Paper II), the hyperphagia than the inclusion of just one unhealthy component (la locus coeruleus (Paper III) and the central amygdala (Paper IV). Fleur et al., 2014). Notably, all available rat atlases are based on mapping of the male brain. Evidence suggests variations in the size of some brain regions, In Paper I, a combination of commercial 60% HFD-pellets and chow such as the medial preoptic area and dorsal locus coeruleus (Babstock were used to induce obesity in mice and rats. In Paper II, mice were et al., 1997; Hofman & Swaab, 1989), between male and female rats. It fed a 60% HFD while rats were offered a free-choice high-fat high- is tempting to question what additional morphological differences sugar diet. In Paper III, the animals were fed a chow-based diet during could be identified, and whether the development of a female rat brain the entire study, except for the initial experiment in which they were atlas could enhance the precision of surgical procedures and collection provided with a free-choice paradigm to test food preference following of micropunches from female brains. a ghrelin injection. In Paper IV we employed Susanne la Fleur’s free- choice paradigm in the initial experiments, and 60% HFD-pellets in Viral vectors the gonadectomized cohort. Viral vectors, such as adeno-associated viruses (AAVs), are essential Surgeries tools for gene delivery in scientific research. In Paper II and IV, AAVs were utilized to knock down the IL-6 gene and CYP19a1 gene, In Paper IV, rats were gonadectomized to investigate the effects of respectively. In both cases this was done with AAVs carrying small reduced estrogen synthesis in amygdala without the compensation of interfering RNA (siRNA) designed to target the messenger RNAs circulating sex hormones sourced from the gonads. Bilateral (mRNA) of interest, induce their degradation and reduce the ovariectomy is the best characterized model for mimicking the human expression of the corresponding gene. This tool is of great value, as it ovarian hormone loss after menopause. In Paper II, telemetric E- enables precise gene targeting in specific regions. It is particularly mitter transponders were implanted in the abdominal cavity of rats. In useful for investigating the physiological significance of a substrate, Paper II, III and IV stereotaxic surgeries were performed to implant such as determining whether the expression of an endogenous peptide, cannulas for drug delivery. Lastly, stereotaxic surgeries were utilized e.g., aromatase, is necessary for normal feeding behavior. Throughout for delivery of viral vectors in Paper II and IV. history, transgenic knockout models have played a pivotal role in unraveling gene function, and knockout mice remain integral to metabolic research (Yazdi et al., 2015). While this approach has been 21 22 Ivana Maric Ivana Maric simple sugars (sucrose) as opposed to regular chow pellets, making All surgeries were performed under ketamine/xylazine anesthesia them more than just high-fat diets. Another vital element in inducing administered intraperitonially. It is common practice in our lab to overeating and emulating the obesogenic conditions in humans is the administer a dosage that is up to 15% lower, per kilogram, to female emphasis on variety, often referred to as the 'buffet effect'. With this rats. However, there is no published data that confirms our observed concept in mind, Susanne la Fleur's laboratory introduced a free-choice sex difference in anesthesia sensitivity. A rat brain atlas (Paxinos & high-fat high-sugar diet, offering lard and a 30% sucrose solution with Watson) was used for decision of appropriate coordinates to target the standard chow. This approach demonstrated a more sustained lateral parabrachial nucleus and paraventricular nucleus (Paper II), the hyperphagia than the inclusion of just one unhealthy component (la locus coeruleus (Paper III) and the central amygdala (Paper IV). Fleur et al., 2014). Notably, all available rat atlases are based on mapping of the male brain. Evidence suggests variations in the size of some brain regions, In Paper I, a combination of commercial 60% HFD-pellets and chow such as the medial preoptic area and dorsal locus coeruleus (Babstock were used to induce obesity in mice and rats. In Paper II, mice were et al., 1997; Hofman & Swaab, 1989), between male and female rats. It fed a 60% HFD while rats were offered a free-choice high-fat high- is tempting to question what additional morphological differences sugar diet. In Paper III, the animals were fed a chow-based diet during could be identified, and whether the development of a female rat brain the entire study, except for the initial experiment in which they were atlas could enhance the precision of surgical procedures and collection provided with a free-choice paradigm to test food preference following of micropunches from female brains. a ghrelin injection. In Paper IV we employed Susanne la Fleur’s free- choice paradigm in the initial experiments, and 60% HFD-pellets in Viral vectors the gonadectomized cohort. Viral vectors, such as adeno-associated viruses (AAVs), are essential Surgeries tools for gene delivery in scientific research. In Paper II and IV, AAVs were utilized to knock down the IL-6 gene and CYP19a1 gene, In Paper IV, rats were gonadectomized to investigate the effects of respectively. In both cases this was done with AAVs carrying small reduced estrogen synthesis in amygdala without the compensation of interfering RNA (siRNA) designed to target the messenger RNAs circulating sex hormones sourced from the gonads. Bilateral (mRNA) of interest, induce their degradation and reduce the ovariectomy is the best characterized model for mimicking the human expression of the corresponding gene. This tool is of great value, as it ovarian hormone loss after menopause. In Paper II, telemetric E- enables precise gene targeting in specific regions. It is particularly mitter transponders were implanted in the abdominal cavity of rats. In useful for investigating the physiological significance of a substrate, Paper II, III and IV stereotaxic surgeries were performed to implant such as determining whether the expression of an endogenous peptide, cannulas for drug delivery. Lastly, stereotaxic surgeries were utilized e.g., aromatase, is necessary for normal feeding behavior. Throughout for delivery of viral vectors in Paper II and IV. history, transgenic knockout models have played a pivotal role in unraveling gene function, and knockout mice remain integral to metabolic research (Yazdi et al., 2015). While this approach has been 21 22 Ivana Maric Ivana Maric invaluable for elucidating the fundamental roles of genes, the newer physiologically 'active' form and extensively studied for its impact on knockdown techniques that we choose to use offer distinct advantages. energy balance. The antagonist employed, LEAP2, represents a In transgenic animals with global null mutations of specific genes, recently identified endogenous antagonist of the GHSR (M'Kadmi et organisms’ risk to develop compensatory mechanisms, complicating al., 2019). Given its novelty, there is currently a scarcity of published the physiological interpretation of the gene. In contrast, AAV- studies concerning the effects of exogenously administered LEAP2 mediated knockdowns in adult animals enable a more direct into discrete brain regions. Notably, as LEAP2 functions as an inverse assessment of the gene’s influence on physiology. We also utilized antagonist, diverging from many previously used synthetic GHSR AAVs for retrograde neural tract tracing. In Paper II the viral vector antagonists, it might unveil fresh insights in the field of ghrelin research AAV2(Retro)-eSyn-EGFP was administered into the PVH, to be taken as its utilization becomes more widespread. up by neurons and transported along their axons to the cell bodies. This technique, together with RNAscope, was used to confirm the To ensure optimal compatibility with the respective injection sites, hypothesis that IL-6 expressing neurons from the lPBN innervate the peptides were dissolved in artificial cerebrospinal fluid (aCSF) for PVH. central administration, and saline solution for peripheral administration. Drugs Temperature and locomotor activity measurements In Paper II, IL-6 and leptin were administered together into the lPBN at subthreshold doses. This approach serves as a method for assessing Locomotor activity and adaptive thermogenesis are two major synergy, and the finding that combining two substances at doses that components of energy expenditure. Several experimental assays are individually ineffective can produce an effect indicates potentially monitor an animal's movements as secondary measure, such as the shared downstream signaling pathways. Gaining insights into the open field test or operant conditioning (as described in Paper I and common downstream signaling pathways can provide a more IV). While these tests can provide insights into the impact of a profound comprehension of the mechanisms driving the observed manipulation on locomotor activity, caution is necessary when effect, offering valuable insights into disease processes and interpreting the results due to the limited duration of these tests and intervention development. Additionally, employing lower doses of confounding factors related to the novelty of the testing environment. each substance to attain the desired effect holds notable To obtain the most accurate measurements of how chronic or acute pharmacological significance and paves the way for investigating novel treatments affect energy expenditure, we employ E-mitter telemetry drug combinations capable of delivering improved therapeutic (Paper II). This approach relies on implants that continuously monitor advantages. locomotor activity and temperature in rats within their home cage. Additionally, we utilize infrared imaging technology (FLIR) to assess To investigate the effects of ghrelin signaling in the LC (Paper III) we temperature in a non-invasive way (Paper I, II and IV). The nature of administered rat ghrelin and LEAP2 centrally. The exogenous ghrelin this technique enables the acquisition of spatial temperature data, used in this study was the acylated form, often regarded the providing an assessment that goes beyond core temperature alone. 23 24 Ivana Maric Ivana Maric invaluable for elucidating the fundamental roles of genes, the newer physiologically 'active' form and extensively studied for its impact on knockdown techniques that we choose to use offer distinct advantages. energy balance. The antagonist employed, LEAP2, represents a In transgenic animals with global null mutations of specific genes, recently identified endogenous antagonist of the GHSR (M'Kadmi et organisms’ risk to develop compensatory mechanisms, complicating al., 2019). Given its novelty, there is currently a scarcity of published the physiological interpretation of the gene. In contrast, AAV- studies concerning the effects of exogenously administered LEAP2 mediated knockdowns in adult animals enable a more direct into discrete brain regions. Notably, as LEAP2 functions as an inverse assessment of the gene’s influence on physiology. We also utilized antagonist, diverging from many previously used synthetic GHSR AAVs for retrograde neural tract tracing. In Paper II the viral vector antagonists, it might unveil fresh insights in the field of ghrelin research AAV2(Retro)-eSyn-EGFP was administered into the PVH, to be taken as its utilization becomes more widespread. up by neurons and transported along their axons to the cell bodies. This technique, together with RNAscope, was used to confirm the To ensure optimal compatibility with the respective injection sites, hypothesis that IL-6 expressing neurons from the lPBN innervate the peptides were dissolved in artificial cerebrospinal fluid (aCSF) for PVH. central administration, and saline solution for peripheral administration. Drugs Temperature and locomotor activity measurements In Paper II, IL-6 and leptin were administered together into the lPBN at subthreshold doses. This approach serves as a method for assessing Locomotor activity and adaptive thermogenesis are two major synergy, and the finding that combining two substances at doses that components of energy expenditure. Several experimental assays are individually ineffective can produce an effect indicates potentially monitor an animal's movements as secondary measure, such as the shared downstream signaling pathways. Gaining insights into the open field test or operant conditioning (as described in Paper I and common downstream signaling pathways can provide a more IV). While these tests can provide insights into the impact of a profound comprehension of the mechanisms driving the observed manipulation on locomotor activity, caution is necessary when effect, offering valuable insights into disease processes and interpreting the results due to the limited duration of these tests and intervention development. Additionally, employing lower doses of confounding factors related to the novelty of the testing environment. each substance to attain the desired effect holds notable To obtain the most accurate measurements of how chronic or acute pharmacological significance and paves the way for investigating novel treatments affect energy expenditure, we employ E-mitter telemetry drug combinations capable of delivering improved therapeutic (Paper II). This approach relies on implants that continuously monitor advantages. locomotor activity and temperature in rats within their home cage. Additionally, we utilize infrared imaging technology (FLIR) to assess To investigate the effects of ghrelin signaling in the LC (Paper III) we temperature in a non-invasive way (Paper I, II and IV). The nature of administered rat ghrelin and LEAP2 centrally. The exogenous ghrelin this technique enables the acquisition of spatial temperature data, used in this study was the acylated form, often regarded the providing an assessment that goes beyond core temperature alone. 23 24 Ivana Maric Ivana Maric However, it's important to note the limitations with this technique, end of the test, the number of lever presses represent how motivated such as potential insulating effects of fur and fat, making it more of a the animal was to obtain the reward. relative measure rather than a precise detection of absolute temperatures. Anxiety-like behavior Operant conditioning The brain regions and circuits that control food intake, reward processing, and motivation are intricately interconnected with those Motivation, ‘wanting’, is an important component of reward, together responsible for emotional regulation. Therefore, it is highly relevant to with hedonic ‘liking’. Food motivation is closely linked to the pleasure include anxiety testing in the research of eating behavior. The and sensory experience associated with eating, and the specific assessment of anxiety-like behavior in rodent models encompasses a incentive value of the food item plays an important role. Nonetheless, range of assays designed to uncover responses that can resemble the in order for food reward to influence eating behavior, pleasure needs intricate human condition of anxiety. to be transformed into motivation Adding to the complexity, while 'liking' and 'wanting' are frequently intertwined, lessons drawn from The open field (Paper II and III) test assesses the animal's exploration substance abuse disorders have shown that motivated reward-seeking of an unfamiliar environment and measures the balance between the behavior can exist in the absence of pleasure. Physiological states like desire to explore a novel space and the anxiety of an open space. In hunger strongly influence the motivation for food, with an increase in the open field (OF) test, locomotion can be an important confounding motivation when hungry and a decrease as satiety is achieved. The factor, particularly when studying both male and female rats. This is brain's mesolimbic pathway, particularly the release of dopamine, have because female rats tend to exhibit higher levels of spontaneous a significant impact on food motivation (Alonso-Alonso et al., 2015). locomotion compared to males. When studying anxiety-like behavior Food cravings are a common manifestation of food motivation, driven in the OF, and other locomotor-based tests such as the elevated plus by sensory cues and emotional states. Taken together, it is clear that maze, the differences in locomotion between sexes or in response to a food motivation is crucial to understand in the study of disordered treatment can complicate the interpretation of results. To mitigate the eating. risk of misinterpretation, we employ an analysis of covariance (ANCOVA) to assess whether the observed effects on anxiety-like Operant conditioning, as studied through the Skinner box paradigm, is behavior remain significant after accounting for differences in a key concept in behavioral neuroscience. In our assay, rats are trained locomotor activity. In addition, we started incorporating alternative to press a lever to obtain a sucrose (Paper III) or fat pellet (Paper tests to more accurately evaluate anxiety-related behaviors in our IV), with the aim to measure the motivation to work for a food reward. animal models. In Paper III, anxiety-like behavior is measured with The progressive ratio (PR) schedule is designed to make the task the acoustic startle response (ASR), a test where the rat is placed in a progressively more challenging as the rat works for each successive confined chamber and exposed to sudden noise bursts. The startle reward. To summarize, this means that the number of lever presses response involves the rapid and involuntary motor response, typically required to obtain each additional sucrose or fat pellet increases. At the a whole-body flinch or jump. A noise burst elicits a startle response 25 26 Ivana Maric Ivana Maric However, it's important to note the limitations with this technique, end of the test, the number of lever presses represent how motivated such as potential insulating effects of fur and fat, making it more of a the animal was to obtain the reward. relative measure rather than a precise detection of absolute temperatures. Anxiety-like behavior Operant conditioning The brain regions and circuits that control food intake, reward processing, and motivation are intricately interconnected with those Motivation, ‘wanting’, is an important component of reward, together responsible for emotional regulation. Therefore, it is highly relevant to with hedonic ‘liking’. Food motivation is closely linked to the pleasure include anxiety testing in the research of eating behavior. The and sensory experience associated with eating, and the specific assessment of anxiety-like behavior in rodent models encompasses a incentive value of the food item plays an important role. Nonetheless, range of assays designed to uncover responses that can resemble the in order for food reward to influence eating behavior, pleasure needs intricate human condition of anxiety. to be transformed into motivation Adding to the complexity, while 'liking' and 'wanting' are frequently intertwined, lessons drawn from The open field (Paper II and III) test assesses the animal's exploration substance abuse disorders have shown that motivated reward-seeking of an unfamiliar environment and measures the balance between the behavior can exist in the absence of pleasure. Physiological states like desire to explore a novel space and the anxiety of an open space. In hunger strongly influence the motivation for food, with an increase in the open field (OF) test, locomotion can be an important confounding motivation when hungry and a decrease as satiety is achieved. The factor, particularly when studying both male and female rats. This is brain's mesolimbic pathway, particularly the release of dopamine, have because female rats tend to exhibit higher levels of spontaneous a significant impact on food motivation (Alonso-Alonso et al., 2015). locomotion compared to males. When studying anxiety-like behavior Food cravings are a common manifestation of food motivation, driven in the OF, and other locomotor-based tests such as the elevated plus by sensory cues and emotional states. Taken together, it is clear that maze, the differences in locomotion between sexes or in response to a food motivation is crucial to understand in the study of disordered treatment can complicate the interpretation of results. To mitigate the eating. risk of misinterpretation, we employ an analysis of covariance (ANCOVA) to assess whether the observed effects on anxiety-like Operant conditioning, as studied through the Skinner box paradigm, is behavior remain significant after accounting for differences in a key concept in behavioral neuroscience. In our assay, rats are trained locomotor activity. In addition, we started incorporating alternative to press a lever to obtain a sucrose (Paper III) or fat pellet (Paper tests to more accurately evaluate anxiety-related behaviors in our IV), with the aim to measure the motivation to work for a food reward. animal models. In Paper III, anxiety-like behavior is measured with The progressive ratio (PR) schedule is designed to make the task the acoustic startle response (ASR), a test where the rat is placed in a progressively more challenging as the rat works for each successive confined chamber and exposed to sudden noise bursts. The startle reward. To summarize, this means that the number of lever presses response involves the rapid and involuntary motor response, typically required to obtain each additional sucrose or fat pellet increases. At the a whole-body flinch or jump. A noise burst elicits a startle response 25 26 Ivana Maric Ivana Maric both in humans and laboratory animals, making this a highly employed to visualize mRNA within brain sections affixed to glass translatable assay (Davis & Whalen, 2001). This response can be slides. This method involves the application of target probes and signal quantified by measuring the magnitude of the motor reaction – a amplifiers, which ultimately generates fluorescent markers for the greater startle response is an indicator of higher vigilance, which is a specific target mRNA that can be imaged. This visual confirmation is fundamental characteristic of anxiety disorders. vital for in pinpointing in what cell type the target gene is expressed. More importantly, it provides the ability to label for several targets, Biochemical procedures enabling the detection of co-expression. In Paper II, RNAscope was employed to detect if IL6 is co-expressed with labels for neurons, For the study of gene expression, frozen brains were sectioned using a microglia and astrocytes in lPBN, to investigate what cell type in this cryostat and micropunches of the PBN (Paper II), LC (Paper III) and brain region is responsible for the production of this cytokine. It was amygdala (Paper IV) were obtained. In Paper I and II, BAT samples also utilized in the retrograde tracing experiment discussed in the were collected and flash frozen during the terminal experiment. The section about viral vectors. In Paper III, GHSR was visualized within fat samples and brain micropunches were processed using a Qiagen the locus coeruleus and confirmed to be co-expressed with TH. Lastly, lipid kit for mRNA extraction, followed by a reverse transcription step in Paper IV, CYP19A1 was visualized throughout neurons in the to synthesize cDNA. Gene expression in the samples was assessed by amygdala, providing support for the capacity to locally produce Quantitative Polymerase Chain Reaction (qPCR) using TaqMan® estrogen. gene expression assays for the target genes. During the qPCR process the template is amplified exponentially over several cycles, and Finally, in Paper II, Western blot was utilized to measure UCP-1 fluorescent signals from the Taqman probe is measured. A highly protein levels in BAT and immunohistochemistry (IHC) was used to expressed gene results in higher fluorescent signal. The number of detect TH in BAT. The Western blot process begins by separating cycles it takes for the fluorescent signal to reach a threshold, will define proteins in a sample based on size using gel electrophoresis, to then the cycle threshold (CT) value for a sample. Analysis of the resulting transfer them to a membrane that is treated with a primary antibody data was then performed using the 2-ΔΔCT method, with beta actin (a targeting the protein of interest, and a secondary antibody that is highly expressed stable gene) as endogenous control gene (Livak & conjugated with a dye allowing quantification. Immunohistochemistry Schmittgen, 2001). This method was used to measure the expression is primarily used for spatial distribution and localization of proteins of genes relevant for BAT thermogenesis in Paper I, to test if they within sections of tissues. Similarly, to the Western blot technique, it were altered in response to diet-induced obesity. In Paper II and IV, relies on primary and secondary antibodies, designed to target the qPCR was utilized to confirm the reduced expression of the genes protein of interest and create a signal that can be imaged. In addition targeted for viral knockdown. Lastly, in Paper III, we analyzed how to visualizing the protein in the tissue, the signals can be quantified GHSR expression in the LC differed between males and females. using imaging processing tools. While qPCR excels at quantitative mRNA analysis, fluorescent in situ To summarize, qPCR and FISH experiments primarily provide hybridization (FISH) techniques like RNAscope adds important information about gene expression at the mRNA level. IHC and nuances to the gene expression data. RNAscope is a technique 27 28 Ivana Maric Ivana Maric both in humans and laboratory animals, making this a highly employed to visualize mRNA within brain sections affixed to glass translatable assay (Davis & Whalen, 2001). This response can be slides. This method involves the application of target probes and signal quantified by measuring the magnitude of the motor reaction – a amplifiers, which ultimately generates fluorescent markers for the greater startle response is an indicator of higher vigilance, which is a specific target mRNA that can be imaged. This visual confirmation is fundamental characteristic of anxiety disorders. vital for in pinpointing in what cell type the target gene is expressed. More importantly, it provides the ability to label for several targets, Biochemical procedures enabling the detection of co-expression. In Paper II, RNAscope was employed to detect if IL6 is co-expressed with labels for neurons, For the study of gene expression, frozen brains were sectioned using a microglia and astrocytes in lPBN, to investigate what cell type in this cryostat and micropunches of the PBN (Paper II), LC (Paper III) and brain region is responsible for the production of this cytokine. It was amygdala (Paper IV) were obtained. In Paper I and II, BAT samples also utilized in the retrograde tracing experiment discussed in the were collected and flash frozen during the terminal experiment. The section about viral vectors. In Paper III, GHSR was visualized within fat samples and brain micropunches were processed using a Qiagen the locus coeruleus and confirmed to be co-expressed with TH. Lastly, lipid kit for mRNA extraction, followed by a reverse transcription step in Paper IV, CYP19A1 was visualized throughout neurons in the to synthesize cDNA. Gene expression in the samples was assessed by amygdala, providing support for the capacity to locally produce Quantitative Polymerase Chain Reaction (qPCR) using TaqMan® estrogen. gene expression assays for the target genes. During the qPCR process the template is amplified exponentially over several cycles, and Finally, in Paper II, Western blot was utilized to measure UCP-1 fluorescent signals from the Taqman probe is measured. A highly protein levels in BAT and immunohistochemistry (IHC) was used to expressed gene results in higher fluorescent signal. The number of detect TH in BAT. The Western blot process begins by separating cycles it takes for the fluorescent signal to reach a threshold, will define proteins in a sample based on size using gel electrophoresis, to then the cycle threshold (CT) value for a sample. Analysis of the resulting transfer them to a membrane that is treated with a primary antibody data was then performed using the 2-ΔΔCT method, with beta actin (a targeting the protein of interest, and a secondary antibody that is highly expressed stable gene) as endogenous control gene (Livak & conjugated with a dye allowing quantification. Immunohistochemistry Schmittgen, 2001). This method was used to measure the expression is primarily used for spatial distribution and localization of proteins of genes relevant for BAT thermogenesis in Paper I, to test if they within sections of tissues. Similarly, to the Western blot technique, it were altered in response to diet-induced obesity. In Paper II and IV, relies on primary and secondary antibodies, designed to target the qPCR was utilized to confirm the reduced expression of the genes protein of interest and create a signal that can be imaged. In addition targeted for viral knockdown. Lastly, in Paper III, we analyzed how to visualizing the protein in the tissue, the signals can be quantified GHSR expression in the LC differed between males and females. using imaging processing tools. While qPCR excels at quantitative mRNA analysis, fluorescent in situ To summarize, qPCR and FISH experiments primarily provide hybridization (FISH) techniques like RNAscope adds important information about gene expression at the mRNA level. IHC and nuances to the gene expression data. RNAscope is a technique 27 28 Ivana Maric Ivana Maric Western blot serve as crucial tools for confirming the presence and RESULTS quantifying the protein products of the studied genes. Integrating these techniques is highly beneficial as it provides a cross-validation mechanism, confirming that alterations at the mRNA level are Paper I genuinely reflected in protein expression changes, which is not always the case. However, limited antibody availability and specificity is an In this work we sought to explore the effect of sex and species in the issue for some proteins like aromatase, causing us to rely primarily on development of diet-induced obesity. Both male and female rats and gene expression quantification. mice were offered a free choice of a 60% HFD and standard chow. Regardless of the species, males demonstrated greater caloric intake Statistical analysis and weight gain when exposed to the HFD. Females displayed a lower level of overeating than males, but exhibited a higher preference for In the studies presented here, data from males and females are the unhealthy diet. While rats preferred the HFD over chow, the consistently examined separately and are never combined. Throughout preference displayed by mice was dramatic, with nearly all of their total majority of the studies, data of both males and females are analyzed intake coming from the palatable diet. Mice showed an inferior ability using a two-factor ANOVA, with sex as one of the variables. A two- to compensate for the higher energy density of the HFD, and they factor ANOVA assesses the influence of two independent variables, consumed a pellet mass similar to controls. Despite male mice gaining here ‘sex’ and ‘treatment/diet’ on a dependent variable. This allowed more weight, it was the females who experienced a remarkable us to not only assess main effects of the independent variables but to accumulation of metabolically unhealthy, visceral fat. Furthermore, the also explore potential interactions. severe level of overeating in mice coincided with compromised energy expenditure, namely reduced levels of locomotor activity and BAT thermogenesis. Collectively, mice of both sexes exhibited a more pronounced obesity phenotype, further evidenced by their disrupted glucose homeostasis. In contrast, obese rats maintained normal glucose tolerance when subjected to an oral glucose challenge. In both rats and mice, females exhibited a higher BAT thermogenesis at baseline compared to males of the same species. However, only female rats displayed a higher locomotor activity than males, potentially contributing to their blunted weight gain. Intriguingly, we found that only male rats increased BAT temperature and expression of genes relevant for thermogenesis in response to the obesogenic diet. 29 30 Ivana Maric Ivana Maric Western blot serve as crucial tools for confirming the presence and RESULTS quantifying the protein products of the studied genes. Integrating these techniques is highly beneficial as it provides a cross-validation mechanism, confirming that alterations at the mRNA level are Paper I genuinely reflected in protein expression changes, which is not always the case. However, limited antibody availability and specificity is an In this work we sought to explore the effect of sex and species in the issue for some proteins like aromatase, causing us to rely primarily on development of diet-induced obesity. Both male and female rats and gene expression quantification. mice were offered a free choice of a 60% HFD and standard chow. Regardless of the species, males demonstrated greater caloric intake Statistical analysis and weight gain when exposed to the HFD. Females displayed a lower level of overeating than males, but exhibited a higher preference for In the studies presented here, data from males and females are the unhealthy diet. While rats preferred the HFD over chow, the consistently examined separately and are never combined. Throughout preference displayed by mice was dramatic, with nearly all of their total majority of the studies, data of both males and females are analyzed intake coming from the palatable diet. Mice showed an inferior ability using a two-factor ANOVA, with sex as one of the variables. A two- to compensate for the higher energy density of the HFD, and they factor ANOVA assesses the influence of two independent variables, consumed a pellet mass similar to controls. Despite male mice gaining here ‘sex’ and ‘treatment/diet’ on a dependent variable. This allowed more weight, it was the females who experienced a remarkable us to not only assess main effects of the independent variables but to accumulation of metabolically unhealthy, visceral fat. Furthermore, the also explore potential interactions. severe level of overeating in mice coincided with compromised energy expenditure, namely reduced levels of locomotor activity and BAT thermogenesis. Collectively, mice of both sexes exhibited a more pronounced obesity phenotype, further evidenced by their disrupted glucose homeostasis. In contrast, obese rats maintained normal glucose tolerance when subjected to an oral glucose challenge. In both rats and mice, females exhibited a higher BAT thermogenesis at baseline compared to males of the same species. However, only female rats displayed a higher locomotor activity than males, potentially contributing to their blunted weight gain. Intriguingly, we found that only male rats increased BAT temperature and expression of genes relevant for thermogenesis in response to the obesogenic diet. 29 30 Ivana Maric Ivana Maric Paper II nearly all retrogradely labeled cells - indicating that lPBN neurons are a source of IL-6 in the PVH. Based on prior discoveries of reduced IL-6 levels in the CSF of obese human patients (Stenlöf et al., 2003), this study opted to examine IL-6 Furthermore, the knockdown animals showed indications of increased gene expression in brain regions key for energy balance in obese male anxiety-like behavior when tested in the OF. To investigate if stress, and female rodents. Among the various brain regions analyzed, the similarly to obesity, could be a source of dysregulated IL-6 in the PBN, PBN emerged as the singular area demonstrating a significant decline we exposed male rats to acute restraint stress. However, this did not in IL-6 gene expression in animals subjected to diet-induced obesity. reveal any alterations in IL-6 gene expression. On the other hand, cold Intriguingly, this was a sex-specific effect only observed in male mice exposure potently increased IL-6 expression in the PBN. Crucially, this and rats. Due to this lack of response of IL-6 in the PBN of females elevation was evident not only in males consuming standard chow, but to any metabolic challenges applied, majority of remaining experiments also in obese males - providing evidence that cold exposure can, to aiming to reveal the mechanism of metabolic effects of IL-6 were some extent, reinstate IL-6 levels in the lPBN that are diminished in performed in male rats only. obesity. Exogenous administration of IL-6 targeting lateral lPBN reduced food intake and produced hyperthermia by significantly increasing BAT temperature. To elucidate the physiological function of IL-6 in the lPBN of male rats, we applied an AAV-mediated knockdown specifically within this brain region, effectively reducing IL-6 expression. Reduction of endogenous IL-6 in the lPBN resulted in body weight gain, that was driven by reduced BAT temperature. Two molecular mechanisms were identified as potential explanations for the attenuated thermogenesis: impaired sympathetic input to BAT and impaired hypothalamus-pituitary-thyroid (HPT) axis. This was characterized by reduced levels of tyrosine hydroxylase (TH) in the BAT of knockdown animals, and a decline in plasma thyroid hormones. Given the regulatory role of the PVH in the HPT axis, we hypothesized that the dysregulation caused by loss of PBN IL-6 may be linked to disrupted signaling to the PVH. To test this hypothesis, we targeted the PVH with a retrograde AAV. The neural tract tracing revealed the presence of retrogradely labeled cell bodies specifically in the lPBN, and more importantly co-expression of IL-6 mRNA on 31 32 Ivana Maric Ivana Maric Paper II nearly all retrogradely labeled cells - indicating that lPBN neurons are a source of IL-6 in the PVH. Based on prior discoveries of reduced IL-6 levels in the CSF of obese human patients (Stenlöf et al., 2003), this study opted to examine IL-6 Furthermore, the knockdown animals showed indications of increased gene expression in brain regions key for energy balance in obese male anxiety-like behavior when tested in the OF. To investigate if stress, and female rodents. Among the various brain regions analyzed, the similarly to obesity, could be a source of dysregulated IL-6 in the PBN, PBN emerged as the singular area demonstrating a significant decline we exposed male rats to acute restraint stress. However, this did not in IL-6 gene expression in animals subjected to diet-induced obesity. reveal any alterations in IL-6 gene expression. On the other hand, cold Intriguingly, this was a sex-specific effect only observed in male mice exposure potently increased IL-6 expression in the PBN. Crucially, this and rats. Due to this lack of response of IL-6 in the PBN of females elevation was evident not only in males consuming standard chow, but to any metabolic challenges applied, majority of remaining experiments also in obese males - providing evidence that cold exposure can, to aiming to reveal the mechanism of metabolic effects of IL-6 were some extent, reinstate IL-6 levels in the lPBN that are diminished in performed in male rats only. obesity. Exogenous administration of IL-6 targeting lateral lPBN reduced food intake and produced hyperthermia by significantly increasing BAT temperature. To elucidate the physiological function of IL-6 in the lPBN of male rats, we applied an AAV-mediated knockdown specifically within this brain region, effectively reducing IL-6 expression. Reduction of endogenous IL-6 in the lPBN resulted in body weight gain, that was driven by reduced BAT temperature. Two molecular mechanisms were identified as potential explanations for the attenuated thermogenesis: impaired sympathetic input to BAT and impaired hypothalamus-pituitary-thyroid (HPT) axis. This was characterized by reduced levels of tyrosine hydroxylase (TH) in the BAT of knockdown animals, and a decline in plasma thyroid hormones. Given the regulatory role of the PVH in the HPT axis, we hypothesized that the dysregulation caused by loss of PBN IL-6 may be linked to disrupted signaling to the PVH. To test this hypothesis, we targeted the PVH with a retrograde AAV. The neural tract tracing revealed the presence of retrogradely labeled cell bodies specifically in the lPBN, and more importantly co-expression of IL-6 mRNA on 31 32 Ivana Maric Ivana Maric Paper III Paper IV Here, we identified the LC as a novel target for the behavioral effects In this study, our primary objective was to investigate the necessity of of ghrelin. First, we established that ghrelin receptors are indeed estrogen synthesis in the brain for the regulation of energy balance. To present in the LC of both male and female rats. Notably, gene achieve this, we conducted experiments with gonadally intact, and expression analysis unveiled remarkable disparities in receptor gonadectomized, males and females. Osmotic pumps were surgically expression between the sexes, with males exhibiting significantly implanted targeting the lateral ventricle, and we proceeded to higher levels than females. This finding raised intriguing questions continuously infuse the aromatase inhibitor Letrozole over a 26-day about potential sex-specific responses to ghrelin signaling in the LC. period. Simultaneously, we closely monitored changes in body weight Exogenous administration of ghrelin targeting the LC stimulated chow and the consumption of a free choice high-fat high-sugar diet, intake in both males and females. While the acute response was similar, including chow, lard, and sucrose. The results yielded intriguing sex a sex-specific delayed effect was notable, with only females consuming differences. Males subjected to Letrozole treatment did not exhibit any more chow after 24 hours. Furthermore, intra-LC ghrelin elevated significant effects on body weight, and consumed less energy than motivated behavior for a sucrose reward in a progressive ratio operant control males. In contrast, females both displayed weight gain in test, with no sex divergence. To explore the impact of blocking ghrelin response to the treatment regardless of hormonal state. Notably, signaling at the level of LC in fasted animals, we utilized the ovariectomized females experienced a more pronounced increase in endogenous GHSR antagonist LEAP2. Intra-LC administration of body weight, coupled with a heightened energy intake. In gonadally LEAP2 reduced food intake in both sexes when offered within one intact females, the weight gain observed was not attributed to increased hour of injection. However, it decreased chow intake in females only food consumption, hinting at changes in energy expenditure as the when administered two hours post-injection. Moreover, in females, underlying cause. A recent imaging study revealed a negative intra-LC microinjection of LEAP2 decreased the motivation for correlation between amygdala aromatase availability and human body sucrose. Although it didn't significantly impact food-seeking behavior, mass index, which prompted us to explore if aromatase in the amygdala locomotor activity was reduced during specific time intervals. In of rats is necessary for energy balance. We utilized a virogenetically- contrast, males displayed no significant changes in food motivated mediated knockdown to investigate the involvement of aromatase in behavior following LC GHSR blockade. Lastly, we wanted to explore the rat amygdala on energy balance and food motivated behavior. if ghrelin's effects in the LC extends to anxiety-like behavior. Intra-LC Amygdala-specific aromatase knockdown did not yield significant ghrelin exhibited anxiolytic effects in females in the ASR test, with alterations in body weight in neither intact nor gonadectomized significantly lower startle amplitudes at the highest sound intensity. animals when fed a chow-only diet. It was in an obesogenic Remarkably, these effects did not manifest in males, as ghrelin environment that females with amygdala aromatase knockdown treatment failed to alter their startle responses in the ASR test. displayed heightened body weight gain and calorie intake. In Conversely, acute pharmacological blockade of LC-GHSR with ovariectomized females, this was accompanied by an enhanced food LEAP2, increased startle response in both male and female rats. motivation, indicating that the overeating was hedonically driven. Surprisingly, amygdala aromatase knockdown led to a reduction in 33 34 Ivana Maric Ivana Maric Paper III Paper IV Here, we identified the LC as a novel target for the behavioral effects In this study, our primary objective was to investigate the necessity of of ghrelin. First, we established that ghrelin receptors are indeed estrogen synthesis in the brain for the regulation of energy balance. To present in the LC of both male and female rats. Notably, gene achieve this, we conducted experiments with gonadally intact, and expression analysis unveiled remarkable disparities in receptor gonadectomized, males and females. Osmotic pumps were surgically expression between the sexes, with males exhibiting significantly implanted targeting the lateral ventricle, and we proceeded to higher levels than females. This finding raised intriguing questions continuously infuse the aromatase inhibitor Letrozole over a 26-day about potential sex-specific responses to ghrelin signaling in the LC. period. Simultaneously, we closely monitored changes in body weight Exogenous administration of ghrelin targeting the LC stimulated chow and the consumption of a free choice high-fat high-sugar diet, intake in both males and females. While the acute response was similar, including chow, lard, and sucrose. The results yielded intriguing sex a sex-specific delayed effect was notable, with only females consuming differences. Males subjected to Letrozole treatment did not exhibit any more chow after 24 hours. Furthermore, intra-LC ghrelin elevated significant effects on body weight, and consumed less energy than motivated behavior for a sucrose reward in a progressive ratio operant control males. In contrast, females both displayed weight gain in test, with no sex divergence. To explore the impact of blocking ghrelin response to the treatment regardless of hormonal state. Notably, signaling at the level of LC in fasted animals, we utilized the ovariectomized females experienced a more pronounced increase in endogenous GHSR antagonist LEAP2. Intra-LC administration of body weight, coupled with a heightened energy intake. In gonadally LEAP2 reduced food intake in both sexes when offered within one intact females, the weight gain observed was not attributed to increased hour of injection. However, it decreased chow intake in females only food consumption, hinting at changes in energy expenditure as the when administered two hours post-injection. Moreover, in females, underlying cause. A recent imaging study revealed a negative intra-LC microinjection of LEAP2 decreased the motivation for correlation between amygdala aromatase availability and human body sucrose. Although it didn't significantly impact food-seeking behavior, mass index, which prompted us to explore if aromatase in the amygdala locomotor activity was reduced during specific time intervals. In of rats is necessary for energy balance. We utilized a virogenetically- contrast, males displayed no significant changes in food motivated mediated knockdown to investigate the involvement of aromatase in behavior following LC GHSR blockade. Lastly, we wanted to explore the rat amygdala on energy balance and food motivated behavior. if ghrelin's effects in the LC extends to anxiety-like behavior. Intra-LC Amygdala-specific aromatase knockdown did not yield significant ghrelin exhibited anxiolytic effects in females in the ASR test, with alterations in body weight in neither intact nor gonadectomized significantly lower startle amplitudes at the highest sound intensity. animals when fed a chow-only diet. It was in an obesogenic Remarkably, these effects did not manifest in males, as ghrelin environment that females with amygdala aromatase knockdown treatment failed to alter their startle responses in the ASR test. displayed heightened body weight gain and calorie intake. In Conversely, acute pharmacological blockade of LC-GHSR with ovariectomized females, this was accompanied by an enhanced food LEAP2, increased startle response in both male and female rats. motivation, indicating that the overeating was hedonically driven. Surprisingly, amygdala aromatase knockdown led to a reduction in 33 34 Ivana Maric Ivana Maric BAT thermogenesis in intact females, while the opposite was true in DISCUSSION ovariectomized females. Ultimately, intact and orchiectomized males displayed no notable effects from the knockdown in any of the The findings presented in Paper I offer valuable insights into the measured parameters. complex interplay between sex, species and the development of diet- induced obesity. It is evident that both sex and species play critical roles in shaping the obesity phenotype and its associated metabolic outcomes. One of the key takeaways is the divergence between species of animal models. The striking difference in dietary preference between rats and mice, with mice almost exclusively consuming the palatable diet, underscores the complexity of species-specific responses to obesogenic diets. This discrepancy calls for caution regarding the generalizability of findings from one species to another and emphasizes the importance of selecting appropriate animal models based on the specific research objectives. It also raises questions about what underlying mechanisms may be causing this divergent response. Considering that HFD mice consumed a pellet mass equivalent to those on a standard diet, it is possible that mice to a greater extent rely more on signals related to stomach distention to terminate consumption of palatable food. The female preference for HFD is intriguing in the light of a recent brain imaging study of obese men and women. They found that women with obesity had alterations in brain signatures associated with emotion-related eating and reward driven eating (Bhatt et al., 2023). The observed compromised energy expenditure in mice, characterized by reduced locomotor activity and diminished BAT thermogenesis, is an intriguing finding of this study. This compromised energy expenditure undoubtedly contributes to the more pronounced obesity phenotype observed in mice. However, our data does not provide conclusive evidence regarding the causal relationship between weight gain and impaired energy expenditure. It remains uncertain whether this dysregulation precedes the onset of morbid obesity, or if it is the result of the extensive fat accumulation causing compromised mobility 35 36 Ivana Maric Ivana Maric BAT thermogenesis in intact females, while the opposite was true in DISCUSSION ovariectomized females. Ultimately, intact and orchiectomized males displayed no notable effects from the knockdown in any of the The findings presented in Paper I offer valuable insights into the measured parameters. complex interplay between sex, species and the development of diet- induced obesity. It is evident that both sex and species play critical roles in shaping the obesity phenotype and its associated metabolic outcomes. One of the key takeaways is the divergence between species of animal models. The striking difference in dietary preference between rats and mice, with mice almost exclusively consuming the palatable diet, underscores the complexity of species-specific responses to obesogenic diets. This discrepancy calls for caution regarding the generalizability of findings from one species to another and emphasizes the importance of selecting appropriate animal models based on the specific research objectives. It also raises questions about what underlying mechanisms may be causing this divergent response. Considering that HFD mice consumed a pellet mass equivalent to those on a standard diet, it is possible that mice to a greater extent rely more on signals related to stomach distention to terminate consumption of palatable food. The female preference for HFD is intriguing in the light of a recent brain imaging study of obese men and women. They found that women with obesity had alterations in brain signatures associated with emotion-related eating and reward driven eating (Bhatt et al., 2023). The observed compromised energy expenditure in mice, characterized by reduced locomotor activity and diminished BAT thermogenesis, is an intriguing finding of this study. This compromised energy expenditure undoubtedly contributes to the more pronounced obesity phenotype observed in mice. However, our data does not provide conclusive evidence regarding the causal relationship between weight gain and impaired energy expenditure. It remains uncertain whether this dysregulation precedes the onset of morbid obesity, or if it is the result of the extensive fat accumulation causing compromised mobility 35 36 Ivana Maric Ivana Maric and increased insulating adipose tissue. However, thermal imaging has which specific environmental factor may have contributed to the less some limitations and the molecular profile of BAT in obese mice is not pronounced overeating observed in the males of Paper II. fully conclusive. Quantification of proteins involved in thermogenesis, Nevertheless, it is imperative to consider this aspect, as it underscores as well as measurement of circulating thyroid hormones, would aid us the notion that varying environmental conditions may yield different in confirming the biological basis of the suspected phenotype. Lower patterns of sex differences. prevalence of BAT has been documented in obese individuals, but it appears that it does not necessarily mean its activity is diminished In Paper II, we conducted an analysis of various brain regions critical (Kulterer et al., 2022). However, also in humans the causal relationship for the regulation of energy in mice and rats exposed to obesity, with in this context remains elusive. In the light of results from Paper II, it a particular focus on changes in IL-6 expression. Here, the PBN stood is plausible that the mice in Paper I developed a more advanced out as the singular area where IL-6 gene expression was significantly obesity, and suffered diminished BAT activity, for instance due to the altered in response to diet-induced obesity. However, this reduction reduction of IL-6 in PBN. Diet-induced thermogenesis has been was observed exclusively in male mice and rats, constituting a clear reported in mice, but with shorter HFD exposure (Essen et al., 2017). sexual dimorphism. The study was motivated by prior observations of In male rats, the extent of overeating reached such staggering reduced IL-6 levels in the cerebrospinal fluid (CSF) of obese human proportions that energy expenditure appeared inadequate, even when patients (Stenlöf et al., 2003). However, it is crucial to emphasize that augmented. However, it is noteworthy that while this sex-specific diet- the human study exclusively featured obese men and the question of induced thermogenesis did not rescue male rats from weight gain, it whether obese women exhibit reduced IL-6 levels in CSF remains appeared to play a role in maintaining relatively healthy glucose unanswered. Nonetheless, the findings of Paper II suggest that obesity tolerance despite an accumulation of unhealthy fat mass might selectively impair brain IL-6 in males. Although the study (Chondronikola et al., 2014). It remains a question whether the BAT successfully demonstrated the necessity of IL-6 within the PBN for activity in rats would subside similarly to what is seen in mice, after normal thermoregulation, it became evident that this was not the case longer exposure to the obesogenic diet. for females as PBN IL-6 levels remained unaltered following metabolic challenges. Should IL-6 or related downstream signaling be pursued as Surprisingly, there is a notable discrepancy in weight gained by males a target for obesity treatment, clinical trials must recognize the and females in Paper I and Paper II, such that males in Paper II potential sex-related disparities. Failure to do so may lead to diluted gained less than their chow-fed counterparts compared to females. The effects in a clinical trial including both men and women, without a rationale behind this discrepancy remains speculative, given the thorough analysis of the sex-specific responses. A recent paper from differing study designs. In Paper II, the animals were individually our laboratory found that IL-6 in the LH mediates food intake in housed and introduced to the obesogenic diet at an earlier age (5 weeks males, but only food motivation in females (López-Ferreras et al., rather than 10 weeks), the diet regimen extended for a longer duration 2021). We did detect IL-6 expression in female PBN, but did not (14 weeks as opposed to 10 weeks), the dietary composition differed pinpoint its role. It calls for investigation if the same neural projections (free access to chow, lard, and sucrose water instead of chow and HFD to the PVH are present in females and whether they serve alternative pellets). Therefore, further investigations are warranted to elucidate functions. It is conceivable that IL-6 in the female PBN exclusively 37 38 Ivana Maric Ivana Maric and increased insulating adipose tissue. However, thermal imaging has which specific environmental factor may have contributed to the less some limitations and the molecular profile of BAT in obese mice is not pronounced overeating observed in the males of Paper II. fully conclusive. Quantification of proteins involved in thermogenesis, Nevertheless, it is imperative to consider this aspect, as it underscores as well as measurement of circulating thyroid hormones, would aid us the notion that varying environmental conditions may yield different in confirming the biological basis of the suspected phenotype. Lower patterns of sex differences. prevalence of BAT has been documented in obese individuals, but it appears that it does not necessarily mean its activity is diminished In Paper II, we conducted an analysis of various brain regions critical (Kulterer et al., 2022). However, also in humans the causal relationship for the regulation of energy in mice and rats exposed to obesity, with in this context remains elusive. In the light of results from Paper II, it a particular focus on changes in IL-6 expression. Here, the PBN stood is plausible that the mice in Paper I developed a more advanced out as the singular area where IL-6 gene expression was significantly obesity, and suffered diminished BAT activity, for instance due to the altered in response to diet-induced obesity. However, this reduction reduction of IL-6 in PBN. Diet-induced thermogenesis has been was observed exclusively in male mice and rats, constituting a clear reported in mice, but with shorter HFD exposure (Essen et al., 2017). sexual dimorphism. The study was motivated by prior observations of In male rats, the extent of overeating reached such staggering reduced IL-6 levels in the cerebrospinal fluid (CSF) of obese human proportions that energy expenditure appeared inadequate, even when patients (Stenlöf et al., 2003). However, it is crucial to emphasize that augmented. However, it is noteworthy that while this sex-specific diet- the human study exclusively featured obese men and the question of induced thermogenesis did not rescue male rats from weight gain, it whether obese women exhibit reduced IL-6 levels in CSF remains appeared to play a role in maintaining relatively healthy glucose unanswered. Nonetheless, the findings of Paper II suggest that obesity tolerance despite an accumulation of unhealthy fat mass might selectively impair brain IL-6 in males. Although the study (Chondronikola et al., 2014). It remains a question whether the BAT successfully demonstrated the necessity of IL-6 within the PBN for activity in rats would subside similarly to what is seen in mice, after normal thermoregulation, it became evident that this was not the case longer exposure to the obesogenic diet. for females as PBN IL-6 levels remained unaltered following metabolic challenges. Should IL-6 or related downstream signaling be pursued as Surprisingly, there is a notable discrepancy in weight gained by males a target for obesity treatment, clinical trials must recognize the and females in Paper I and Paper II, such that males in Paper II potential sex-related disparities. Failure to do so may lead to diluted gained less than their chow-fed counterparts compared to females. The effects in a clinical trial including both men and women, without a rationale behind this discrepancy remains speculative, given the thorough analysis of the sex-specific responses. A recent paper from differing study designs. In Paper II, the animals were individually our laboratory found that IL-6 in the LH mediates food intake in housed and introduced to the obesogenic diet at an earlier age (5 weeks males, but only food motivation in females (López-Ferreras et al., rather than 10 weeks), the diet regimen extended for a longer duration 2021). We did detect IL-6 expression in female PBN, but did not (14 weeks as opposed to 10 weeks), the dietary composition differed pinpoint its role. It calls for investigation if the same neural projections (free access to chow, lard, and sucrose water instead of chow and HFD to the PVH are present in females and whether they serve alternative pellets). Therefore, further investigations are warranted to elucidate functions. It is conceivable that IL-6 in the female PBN exclusively 37 38 Ivana Maric Ivana Maric plays a role in regulating food-motivated behavior as is the case in the other sites than LC with GHSR-TH co-expression, such as the NTS LH. Nevertheless, our study did not examine this aspect, nor did we and VTA, and these have been shown to mediate ghrelin’s orexigenic investigate the impact of knockdown in females. It is also plausible that effects, however here we show a GHSR-TH population that also alters PBN IL-6 in females might be implicated in stress regulation, despite anxiety-like behavior. Furthermore, Chuang and colleagues' research, the minimal relevance for the measured parameters in males. Prior along with the majority of other studies examining the impact of research has revealed a sex-specific influence of IL-6 on stress ghrelin on anxiety-like behavior, predominantly involve male animals. responses. In this context, female mice show an elevated Considering the sexually divergent response of LC to stress, it surely corticosterone response to restraint stress, and this distinction calls for investigation to see how ghrelin can mitigate effects following diminishes in the absence of IL-6. While IL-6 knockout female mice stress-exposure also in females. have a reduced HPA activation in response to stress, the response in IL-6 KO males remain similar to wild type (Bethin et al., 2000). In Paper IV, we demonstrate that inhibiting estrogen synthesis specifically in the brain is sufficient to induce sex-specific changes in Prevalence in stress associated pathologies, such as anxiety disorders, feeding behavior. We further establish that estrogen production within are overrepresented in women (Altemus et al., 2014). The findings the amygdala is crucial for maintaining normal thermoregulation and presented in Paper III further extend our understanding of ghrelin's feeding behavior in an obesogenic environment, particularly in impact on the interplay between feeding behavior and emotionality. ovariectomized females. Notably, a recent human study has shown a While a prior study suggested the locus coeruleus (LC) as a potential negative correlation between the amount of amygdala aromatase and location for ghrelin binding, the impact of ghrelin signaling at this site body weight, bolstering the translational relevance of our findings and has remained unexplored until now (Cabral et al., 2013). The divergent suggesting a potential causal link between the two factors (Biegon et outcomes observed between sexes invite two essential questions: is the al., 2020). While reduced estrogen levels are the hypothesized cause for sensitivity to dysregulation, for instance by stress, in this system the effects following loss of aromatase, we must also consider the sexually divergent, and could this system be more effectively targeted potential change in substrate, specifically testosterone, as a for treating anxiety in one sex over the other? Exploring the cellular contributing element. Moreover, aromatase activity in the amygdala profile of GHSR expressing cells in the LC merits attention. does not necessarily mean a local release of estrogen. Hence, we aim Furthermore, discerning the involvement of norepinephrine as a to investigate where these cells project and what their downstream downstream mediator of ghrelin will provide valuable insights into the targets may be. Intriguingly, the presence of aromatase in this brain regulatory mechanisms at play. GHSR KO mice have repeatedly been region in males raises questions about its function at this site. Unlike shown to exhibit increased anxiety-like behavior, and one previous our findings, the human imaging study did not identify sex differences study suggested an obligatory role for GHSR expressed specifically in in the correlation between BMI and amygdala aromatase activity. catecholaminergic neurons in mediating stress-induced overeating, antidepressant-like behavior and food reward (Chuang et al., 2011). The implications of the findings in Paper IV emphasize the The paper did not elucidate what catecholaminergic sites that could be importance of considering not only peripheral estrogen but also the responsible for these effects, but LC is a plausible candidate. There are brain-derived sources of estradiol in future investigations related to the 39 40 Ivana Maric Ivana Maric plays a role in regulating food-motivated behavior as is the case in the other sites than LC with GHSR-TH co-expression, such as the NTS LH. Nevertheless, our study did not examine this aspect, nor did we and VTA, and these have been shown to mediate ghrelin’s orexigenic investigate the impact of knockdown in females. It is also plausible that effects, however here we show a GHSR-TH population that also alters PBN IL-6 in females might be implicated in stress regulation, despite anxiety-like behavior. Furthermore, Chuang and colleagues' research, the minimal relevance for the measured parameters in males. Prior along with the majority of other studies examining the impact of research has revealed a sex-specific influence of IL-6 on stress ghrelin on anxiety-like behavior, predominantly involve male animals. responses. In this context, female mice show an elevated Considering the sexually divergent response of LC to stress, it surely corticosterone response to restraint stress, and this distinction calls for investigation to see how ghrelin can mitigate effects following diminishes in the absence of IL-6. While IL-6 knockout female mice stress-exposure also in females. have a reduced HPA activation in response to stress, the response in IL-6 KO males remain similar to wild type (Bethin et al., 2000). In Paper IV, we demonstrate that inhibiting estrogen synthesis specifically in the brain is sufficient to induce sex-specific changes in Prevalence in stress associated pathologies, such as anxiety disorders, feeding behavior. We further establish that estrogen production within are overrepresented in women (Altemus et al., 2014). The findings the amygdala is crucial for maintaining normal thermoregulation and presented in Paper III further extend our understanding of ghrelin's feeding behavior in an obesogenic environment, particularly in impact on the interplay between feeding behavior and emotionality. ovariectomized females. Notably, a recent human study has shown a While a prior study suggested the locus coeruleus (LC) as a potential negative correlation between the amount of amygdala aromatase and location for ghrelin binding, the impact of ghrelin signaling at this site body weight, bolstering the translational relevance of our findings and has remained unexplored until now (Cabral et al., 2013). The divergent suggesting a potential causal link between the two factors (Biegon et outcomes observed between sexes invite two essential questions: is the al., 2020). While reduced estrogen levels are the hypothesized cause for sensitivity to dysregulation, for instance by stress, in this system the effects following loss of aromatase, we must also consider the sexually divergent, and could this system be more effectively targeted potential change in substrate, specifically testosterone, as a for treating anxiety in one sex over the other? Exploring the cellular contributing element. Moreover, aromatase activity in the amygdala profile of GHSR expressing cells in the LC merits attention. does not necessarily mean a local release of estrogen. Hence, we aim Furthermore, discerning the involvement of norepinephrine as a to investigate where these cells project and what their downstream downstream mediator of ghrelin will provide valuable insights into the targets may be. Intriguingly, the presence of aromatase in this brain regulatory mechanisms at play. GHSR KO mice have repeatedly been region in males raises questions about its function at this site. Unlike shown to exhibit increased anxiety-like behavior, and one previous our findings, the human imaging study did not identify sex differences study suggested an obligatory role for GHSR expressed specifically in in the correlation between BMI and amygdala aromatase activity. catecholaminergic neurons in mediating stress-induced overeating, antidepressant-like behavior and food reward (Chuang et al., 2011). The implications of the findings in Paper IV emphasize the The paper did not elucidate what catecholaminergic sites that could be importance of considering not only peripheral estrogen but also the responsible for these effects, but LC is a plausible candidate. There are brain-derived sources of estradiol in future investigations related to the 39 40 Ivana Maric Ivana Maric effects of sex hormones on energy balance. Regarding IL-6, we've Acknowledgements gained insights into how peripheral and central sources can yield distinct effects. Similarly, when examining aromatase, it is essential to There are many people without whom the work presented here would not investigate how the brain-specific pool is modified in be possible. I would like to express my deepest appreciation to my pathophysiological contexts. supervisor, Karolina Skibicka, who has given me this opportunity and dedicated much time, effort and knowledge to aid me in this journey. Thank you for your patience, guidance and enthusiasm. I would also like Importantly, our work may contribute to insights for the clinical use of to thank the past and present members of the Skibicka lab that I have had aromatase inhibitors. This drug class is associated with side effects like the pleasure to work with. Special thanks to my peers Mohammed and weight gain and hot flashes (Rand et al., 2011). Our results propose Stina, with whom I have shared laughter, frustration and companionship, that these side effects may stem from central actions of aromatase and Jean-Philippe and Francesco for providing valuable advice and inhibition, rather than peripheral actions. It is tempting to theorize that expertise. A heartfelt thank you to Milica for her selfless support, especially for the generous assistance with the Serbian summary and during these side effects could be prevented by limiting the drug’s entry into the final experiment of my debut project. I will miss the enjoyable coffee the brain, thereby ensuring it primarily targets peripheral sites. This breaks with department colleagues, especially Sansan, whose kindness I approach aligns with ongoing research involving compounds like greatly appreciated, and Paul, whose humor was a true mood lifter during oxytocin and GLP-1 analogs, where drug molecules are linked to more lab days. complex organic structures to prevent central side effects (Asker et al., I am also indebted to all my friends for their love, patience and 2023; Borner et al., 2020). Finally, our results hold translational camaraderie. Olle, I would not have made it this far without your significance, as unpublished findings suggest that aromatase inhibitors friendship. Julia, it is hard to express my admiration and appreciation for contribute to weight gain in patients and reduce the effectiveness of you, both during my time at Penn State and beyond. Amanda, I feel weight loss medications among breast cancer survivors (Endocrine fortunate to have had you by my side since we were kids. Olesya, Daniele Society, 2023). and Jose, I am grateful for your loving presence, whether as a comforting shoulder to lean on or as joyful drinking companions. Nick and the Chermols, I cannot thank you enough for making me feel like family; our shared moments will forever hold a special place in my heart. Cecilia, Ellen, Emma and Sofija, I am grateful for your unwavering care, and for sticking around despite my periods of absence. Carl, your encouragement played a vital role in the dissertation writing process. Lastly, I cannot begin to express my gratitude to my family for all of their unconditional love, support, understanding and encouragement. I would like to thank my devoted parents, Vesna and Dragisa, for always being there for me, and my cherished siblings, Milan and Anastasija, for always cheering me on. All my accomplishments, I owe to you. 41 42 Ivana Maric Ivana Maric effects of sex hormones on energy balance. Regarding IL-6, we've Acknowledgements gained insights into how peripheral and central sources can yield distinct effects. Similarly, when examining aromatase, it is essential to There are many people without whom the work presented here would not investigate how the brain-specific pool is modified in be possible. I would like to express my deepest appreciation to my pathophysiological contexts. supervisor, Karolina Skibicka, who has given me this opportunity and dedicated much time, effort and knowledge to aid me in this journey. Thank you for your patience, guidance and enthusiasm. I would also like Importantly, our work may contribute to insights for the clinical use of to thank the past and present members of the Skibicka lab that I have had aromatase inhibitors. This drug class is associated with side effects like the pleasure to work with. Special thanks to my peers Mohammed and weight gain and hot flashes (Rand et al., 2011). Our results propose Stina, with whom I have shared laughter, frustration and companionship, that these side effects may stem from central actions of aromatase and Jean-Philippe and Francesco for providing valuable advice and inhibition, rather than peripheral actions. It is tempting to theorize that expertise. A heartfelt thank you to Milica for her selfless support, especially for the generous assistance with the Serbian summary and during these side effects could be prevented by limiting the drug’s entry into the final experiment of my debut project. I will miss the enjoyable coffee the brain, thereby ensuring it primarily targets peripheral sites. This breaks with department colleagues, especially Sansan, whose kindness I approach aligns with ongoing research involving compounds like greatly appreciated, and Paul, whose humor was a true mood lifter during oxytocin and GLP-1 analogs, where drug molecules are linked to more lab days. complex organic structures to prevent central side effects (Asker et al., I am also indebted to all my friends for their love, patience and 2023; Borner et al., 2020). Finally, our results hold translational camaraderie. Olle, I would not have made it this far without your significance, as unpublished findings suggest that aromatase inhibitors friendship. Julia, it is hard to express my admiration and appreciation for contribute to weight gain in patients and reduce the effectiveness of you, both during my time at Penn State and beyond. Amanda, I feel weight loss medications among breast cancer survivors (Endocrine fortunate to have had you by my side since we were kids. Olesya, Daniele Society, 2023). and Jose, I am grateful for your loving presence, whether as a comforting shoulder to lean on or as joyful drinking companions. Nick and the Chermols, I cannot thank you enough for making me feel like family; our shared moments will forever hold a special place in my heart. Cecilia, Ellen, Emma and Sofija, I am grateful for your unwavering care, and for sticking around despite my periods of absence. Carl, your encouragement played a vital role in the dissertation writing process. Lastly, I cannot begin to express my gratitude to my family for all of their unconditional love, support, understanding and encouragement. I would like to thank my devoted parents, Vesna and Dragisa, for always being there for me, and my cherished siblings, Milan and Anastasija, for always cheering me on. All my accomplishments, I owe to you. 41 42 Ivana Maric Ivana Maric References Abizaid, A., & Horvath, T. L. (2012). Ghrelin and the central regulation of feeding and energy balance. Indian J Endocrinol Metab, 16(Suppl 3), S617-626. https://doi.org/10.4103/2230-8210.105580 Alhadeff, A. L., Hayes, M. R., & Grill, H. J. (2014). Leptin receptor signaling in the lateral parabrachial nucleus contributes to the control of food intake. Am J Physiol Regul Integr Comp Physiol, 307(11), R1338-1344. https://doi.org/10.1152/ajpregu.00329.2014 Alhadeff, A. L., Rupprecht, L. E., & Hayes, M. R. (2012). GLP-1 Neurons in the Nucleus of the Solitary Tract Project Directly to the Ventral Tegmental Area and Nucleus Accumbens to Control for Food Intake. Endocrinology, 153(2), 647-658. https://doi.org/10.1210/en.2011-1443 Alonso-Alonso, M., Woods, S. C., Pelchat, M., Grigson, P. S., Stice, E., Farooqi, S., . . . Beauchamp, G. K. (2015). Food reward system: current perspectives and future research needs. Nutrition Reviews, 73(5), 296-307. https://doi.org/10.1093/nutrit/nuv002 Altemus, M., Sarvaiya, N., & Neill Epperson, C. (2014). Sex differences in anxiety and depression clinical perspectives. Front Neuroendocrinol, 35(3), 320-330. https://doi.org/10.1016/j.yfrne.2014.05.004 Alvarez-Crespo, M., Skibicka, K. P., Farkas, I., Molnár, C. S., Egecioglu, E., Hrabovszky, E., . . . Dickson, S. L. (2012). The amygdala as a neurobiological target for ghrelin in rats: neuroanatomical, electrophysiological and behavioral evidence. Anand, B. K., & Brobeck, J. R. (1951). Localization of a "feeding center" in the hypothalamus of the rat. Proc Soc Exp Biol Med, 77(2), 323-324. https://doi.org/10.3181/00379727-77-18766 Arnold, A. P., & Breedlove, S. M. (1985). Organizational and activational effects of sex steroids on brain and behavior: A reanalysis. Hormones and Behavior, 19(4), 469-498. https://doi.org/10.1016/0018-506X(85)90042-X Asarian, L., & Geary, N. (2002). Cyclic estradiol treatment normalizes body weight and restores physiological patterns of spontaneous feeding and sexual receptivity in ovariectomized rats. Horm Behav, 42(4), 461-471. https://doi.org/10.1006/hbeh.2002.1835 Asker, M., Krieger, J. P., Liles, A., Tinsley, I. C., Borner, T., Maric, I., . . . Skibicka, K. P. (2023). Peripherally restricted oxytocin is sufficient to reduce food intake and motivation, while CNS entry is required for locomotor and taste avoidance effects. Diabetes Obes Metab, 25(3), 856-877. https://doi.org/10.1111/dom.14937 Babstock, D., Malsbury, C. W., & Harley, C. W. (1997). The dorsal locus coeruleus is larger in male than in female Sprague–Dawley rats. Neuroscience Letters, 224(3), 157-160. https://doi.org/https://doi.org/10.1016/S0304- 3940(97)13462-0 43 44 Ivana Maric Ivana Maric References Abizaid, A., & Horvath, T. L. (2012). Ghrelin and the central regulation of feeding and energy balance. Indian J Endocrinol Metab, 16(Suppl 3), S617-626. https://doi.org/10.4103/2230-8210.105580 Alhadeff, A. L., Hayes, M. R., & Grill, H. J. (2014). Leptin receptor signaling in the lateral parabrachial nucleus contributes to the control of food intake. Am J Physiol Regul Integr Comp Physiol, 307(11), R1338-1344. https://doi.org/10.1152/ajpregu.00329.2014 Alhadeff, A. L., Rupprecht, L. E., & Hayes, M. R. (2012). GLP-1 Neurons in the Nucleus of the Solitary Tract Project Directly to the Ventral Tegmental Area and Nucleus Accumbens to Control for Food Intake. Endocrinology, 153(2), 647-658. https://doi.org/10.1210/en.2011-1443 Alonso-Alonso, M., Woods, S. C., Pelchat, M., Grigson, P. S., Stice, E., Farooqi, S., . . . Beauchamp, G. K. (2015). Food reward system: current perspectives and future research needs. Nutrition Reviews, 73(5), 296-307. https://doi.org/10.1093/nutrit/nuv002 Altemus, M., Sarvaiya, N., & Neill Epperson, C. (2014). Sex differences in anxiety and depression clinical perspectives. Front Neuroendocrinol, 35(3), 320-330. https://doi.org/10.1016/j.yfrne.2014.05.004 Alvarez-Crespo, M., Skibicka, K. P., Farkas, I., Molnár, C. S., Egecioglu, E., Hrabovszky, E., . . . Dickson, S. L. (2012). The amygdala as a neurobiological target for ghrelin in rats: neuroanatomical, electrophysiological and behavioral evidence. Anand, B. K., & Brobeck, J. R. (1951). Localization of a "feeding center" in the hypothalamus of the rat. Proc Soc Exp Biol Med, 77(2), 323-324. https://doi.org/10.3181/00379727-77-18766 Arnold, A. P., & Breedlove, S. M. (1985). Organizational and activational effects of sex steroids on brain and behavior: A reanalysis. Hormones and Behavior, 19(4), 469-498. https://doi.org/10.1016/0018-506X(85)90042-X Asarian, L., & Geary, N. (2002). Cyclic estradiol treatment normalizes body weight and restores physiological patterns of spontaneous feeding and sexual receptivity in ovariectomized rats. Horm Behav, 42(4), 461-471. https://doi.org/10.1006/hbeh.2002.1835 Asker, M., Krieger, J. P., Liles, A., Tinsley, I. C., Borner, T., Maric, I., . . . Skibicka, K. P. (2023). Peripherally restricted oxytocin is sufficient to reduce food intake and motivation, while CNS entry is required for locomotor and taste avoidance effects. Diabetes Obes Metab, 25(3), 856-877. https://doi.org/10.1111/dom.14937 Babstock, D., Malsbury, C. W., & Harley, C. W. (1997). The dorsal locus coeruleus is larger in male than in female Sprague–Dawley rats. Neuroscience Letters, 224(3), 157-160. https://doi.org/https://doi.org/10.1016/S0304- 3940(97)13462-0 43 44 Ivana Maric Ivana Maric Bangasser, D. A., Curtis, A., Reyes, B. A., Bethea, T. T., Parastatidis, I., axis during immune system activation. Proc Natl Acad Sci U S A, 97(16), Ischiropoulos, H., . . . Valentino, R. J. (2010). Sex differences in 9317-9322. https://doi.org/10.1073/pnas.97.16.9317 corticotropin-releasing factor receptor signaling and trafficking: potential Bhatt, R. R., Todorov, S., Sood, R., Ravichandran, S., Kilpatrick, L. A., Peng, N., role in female vulnerability to stress-related psychopathology. Mol . . . Gupta, A. (2023). Integrated multi-modal brain signatures predict sex- Psychiatry, 15(9), 877, 896-904. https://doi.org/10.1038/mp.2010.66 specific obesity status. Brain Communications, 5(2), fcad098. Bangasser, D. A., Reyes, B. A., Piel, D., Garachh, V., Zhang, X. Y., Plona, Z. M., https://doi.org/10.1093/braincomms/fcad098 . . . Valentino, R. J. (2013). Increased vulnerability of the brain Biegon, A. (2016). In vivo visualization of aromatase in animals and humans. Front norepinephrine system of females to corticotropin-releasing factor Neuroendocrinol, 40, 42-51. https://doi.org/10.1016/j.yfrne.2015.10.001 overexpression. Mol Psychiatry, 18(2), 166-173. Biegon, A., Alia-Klein, N., Alexoff, D. L., Fowler, J. S., Kim, S. W., Logan, J., . . https://doi.org/10.1038/mp.2012.24 . Hildebrandt, T. (2020). Relationship of estrogen synthesis capacity in Bangasser, D. A., Wiersielis, K. R., & Khantsis, S. (2016). Sex differences in the the brain with obesity and self-control in men and women. Proc Natl Acad locus coeruleus-norepinephrine system and its regulation by stress. Brain Sci U S A, 117(37), 22962-22966. Res, 1641(Pt B), 177-188. https://doi.org/10.1073/pnas.2006117117 https://doi.org/10.1016/j.brainres.2015.11.021 Borner, T., Shaulson, E. D., Tinsley, I. C., Stein, L. M., Horn, C. C., Hayes, M. R., Bapat, S. P., Whitty, C., Mowery, C. T., Liang, Y., Yoo, A., Jiang, Z., . . . Marson, . . . De Jonghe, B. C. (2020). A second-generation glucagon-like peptide- A. (2022). Obesity alters pathology and treatment response in 1 receptor agonist mitigates vomiting and anorexia while retaining inflammatory disease. Nature, 604(7905), 337-342. glucoregulatory potency in lean diabetic and emetic mammalian models. https://doi.org/10.1038/s41586-022-04536-0 Diabetes Obes Metab, 22(10), 1729-1741. Bardo, M. T., Donohew, R. L., & Harrington, N. G. (1996). Psychobiology of https://doi.org/10.1111/dom.14089 novelty seeking and drug seeking behavior. Behavioural Brain Research, Buczek, L., Migliaccio, J., & Petrovich, G. D. (2020). Hedonic Eating: Sex 77(1), 23-43. https://doi.org/https://doi.org/10.1016/0166- Differences and Characterization of Orexin Activation and Signaling. 4328(95)00203-0 Neuroscience, 436, 34-45. Barkan, A. L., Dimaraki, E. V., Jessup, S. K., Symons, K. V., Ermolenko, M., & https://doi.org/10.1016/j.neuroscience.2020.04.008 Jaffe, C. A. (2003). Ghrelin secretion in humans is sexually dimorphic, Börchers, S., Krieger, J. P., Maric, I., Carl, J., Abraham, M., Longo, F., . . . Skibicka, suppressed by somatostatin, and not affected by the ambient growth K. P. (2022). From an Empty Stomach to Anxiolysis: Molecular and hormone levels. J Clin Endocrinol Metab, 88(5), 2180-2184. Behavioral Assessment of Sex Differences in the Ghrelin Axis of Rats. https://doi.org/10.1210/jc.2002-021169 Front Endocrinol (Lausanne), 13, 901669. Becker, J. B., Prendergast, B. J., & Liang, J. W. (2016). Female rats are not more https://doi.org/10.3389/fendo.2022.901669 variable than male rats: a meta-analysis of neuroscience studies. Biol Sex Cabral, A., De Francesco, P. N., & Perello, M. (2015). Brain circuits mediating the Differ, 7, 34. https://doi.org/10.1186/s13293-016-0087-5 orexigenic action of peripheral ghrelin: narrow gates for a vast kingdom. Beery, A. K., & Zucker, I. (2011). Sex bias in neuroscience and biomedical Front Endocrinol (Lausanne), 6, 44. research. Neurosci Biobehav Rev, 35(3), 565-572. https://doi.org/10.3389/fendo.2015.00044 https://doi.org/10.1016/j.neubiorev.2010.07.002 Cabral, A., Fernandez, G., & Perello, M. (2013). Analysis of brain nuclei accessible Benrick, A., Schéle, E., Pinnock, S. B., Wernstedt-Asterholm, I., Dickson, S. L., to ghrelin present in the cerebrospinal fluid. Neuroscience, 253, 406-415. Karlsson-Lindahl, L., & Jansson, J. O. (2009). Interleukin-6 gene https://doi.org/10.1016/j.neuroscience.2013.09.008 knockout influences energy balance regulating peptides in the Cabral, A., Portiansky, E., Sánchez-Jaramillo, E., Zigman, J. M., & Perello, M. hypothalamic paraventricular and supraoptic nuclei. J Neuroendocrinol, (2016). Ghrelin activates hypophysiotropic corticotropin-releasing factor 21(7), 620-628. https://doi.org/10.1111/j.1365-2826.2009.01879.x neurons independently of the arcuate nucleus. Psychoneuroendocrinology, 67, Benz, V., Bloch, M., Wardat, S., Böhm, C., Maurer, L., Mahmoodzadeh, S., . . . 27-39. https://doi.org/10.1016/j.psyneuen.2016.01.027 Kintscher, U. (2012). Sexual dimorphic regulation of body weight Callard, G. V., Petro, Z., & Ryan, K. J. (1978). Phylogenetic Distribution of dynamics and adipose tissue lipolysis. PLoS One, 7(5), e37794. Aromatase and Other Androgen-Converting Enzymes in the Central https://doi.org/10.1371/journal.pone.0037794 Nervous System*. Endocrinology, 103(6), 2283-2290. Bethin, K. E., Vogt, S. K., & Muglia, L. J. (2000). Interleukin-6 is an essential, https://doi.org/10.1210/endo-103-6-2283 corticotropin-releasing hormone-independent stimulator of the adrenal 45 46 Ivana Maric Ivana Maric Bangasser, D. A., Curtis, A., Reyes, B. A., Bethea, T. T., Parastatidis, I., axis during immune system activation. Proc Natl Acad Sci U S A, 97(16), Ischiropoulos, H., . . . Valentino, R. J. (2010). Sex differences in 9317-9322. https://doi.org/10.1073/pnas.97.16.9317 corticotropin-releasing factor receptor signaling and trafficking: potential Bhatt, R. R., Todorov, S., Sood, R., Ravichandran, S., Kilpatrick, L. A., Peng, N., role in female vulnerability to stress-related psychopathology. Mol . . . Gupta, A. (2023). Integrated multi-modal brain signatures predict sex- Psychiatry, 15(9), 877, 896-904. https://doi.org/10.1038/mp.2010.66 specific obesity status. Brain Communications, 5(2), fcad098. Bangasser, D. A., Reyes, B. A., Piel, D., Garachh, V., Zhang, X. Y., Plona, Z. M., https://doi.org/10.1093/braincomms/fcad098 . . . Valentino, R. J. (2013). Increased vulnerability of the brain Biegon, A. (2016). In vivo visualization of aromatase in animals and humans. Front norepinephrine system of females to corticotropin-releasing factor Neuroendocrinol, 40, 42-51. https://doi.org/10.1016/j.yfrne.2015.10.001 overexpression. Mol Psychiatry, 18(2), 166-173. Biegon, A., Alia-Klein, N., Alexoff, D. L., Fowler, J. S., Kim, S. W., Logan, J., . . https://doi.org/10.1038/mp.2012.24 . Hildebrandt, T. (2020). Relationship of estrogen synthesis capacity in Bangasser, D. A., Wiersielis, K. R., & Khantsis, S. (2016). Sex differences in the the brain with obesity and self-control in men and women. Proc Natl Acad locus coeruleus-norepinephrine system and its regulation by stress. Brain Sci U S A, 117(37), 22962-22966. Res, 1641(Pt B), 177-188. https://doi.org/10.1073/pnas.2006117117 https://doi.org/10.1016/j.brainres.2015.11.021 Borner, T., Shaulson, E. D., Tinsley, I. C., Stein, L. M., Horn, C. C., Hayes, M. R., Bapat, S. P., Whitty, C., Mowery, C. T., Liang, Y., Yoo, A., Jiang, Z., . . . Marson, . . . De Jonghe, B. C. (2020). A second-generation glucagon-like peptide- A. (2022). Obesity alters pathology and treatment response in 1 receptor agonist mitigates vomiting and anorexia while retaining inflammatory disease. Nature, 604(7905), 337-342. glucoregulatory potency in lean diabetic and emetic mammalian models. https://doi.org/10.1038/s41586-022-04536-0 Diabetes Obes Metab, 22(10), 1729-1741. Bardo, M. T., Donohew, R. L., & Harrington, N. G. (1996). Psychobiology of https://doi.org/10.1111/dom.14089 novelty seeking and drug seeking behavior. Behavioural Brain Research, Buczek, L., Migliaccio, J., & Petrovich, G. D. (2020). Hedonic Eating: Sex 77(1), 23-43. https://doi.org/https://doi.org/10.1016/0166- Differences and Characterization of Orexin Activation and Signaling. 4328(95)00203-0 Neuroscience, 436, 34-45. Barkan, A. L., Dimaraki, E. V., Jessup, S. K., Symons, K. V., Ermolenko, M., & https://doi.org/10.1016/j.neuroscience.2020.04.008 Jaffe, C. A. (2003). Ghrelin secretion in humans is sexually dimorphic, Börchers, S., Krieger, J. P., Maric, I., Carl, J., Abraham, M., Longo, F., . . . Skibicka, suppressed by somatostatin, and not affected by the ambient growth K. P. (2022). From an Empty Stomach to Anxiolysis: Molecular and hormone levels. J Clin Endocrinol Metab, 88(5), 2180-2184. Behavioral Assessment of Sex Differences in the Ghrelin Axis of Rats. https://doi.org/10.1210/jc.2002-021169 Front Endocrinol (Lausanne), 13, 901669. Becker, J. B., Prendergast, B. J., & Liang, J. W. (2016). Female rats are not more https://doi.org/10.3389/fendo.2022.901669 variable than male rats: a meta-analysis of neuroscience studies. Biol Sex Cabral, A., De Francesco, P. N., & Perello, M. (2015). Brain circuits mediating the Differ, 7, 34. https://doi.org/10.1186/s13293-016-0087-5 orexigenic action of peripheral ghrelin: narrow gates for a vast kingdom. Beery, A. K., & Zucker, I. (2011). Sex bias in neuroscience and biomedical Front Endocrinol (Lausanne), 6, 44. research. Neurosci Biobehav Rev, 35(3), 565-572. https://doi.org/10.3389/fendo.2015.00044 https://doi.org/10.1016/j.neubiorev.2010.07.002 Cabral, A., Fernandez, G., & Perello, M. (2013). Analysis of brain nuclei accessible Benrick, A., Schéle, E., Pinnock, S. B., Wernstedt-Asterholm, I., Dickson, S. L., to ghrelin present in the cerebrospinal fluid. Neuroscience, 253, 406-415. Karlsson-Lindahl, L., & Jansson, J. O. (2009). Interleukin-6 gene https://doi.org/10.1016/j.neuroscience.2013.09.008 knockout influences energy balance regulating peptides in the Cabral, A., Portiansky, E., Sánchez-Jaramillo, E., Zigman, J. M., & Perello, M. hypothalamic paraventricular and supraoptic nuclei. J Neuroendocrinol, (2016). Ghrelin activates hypophysiotropic corticotropin-releasing factor 21(7), 620-628. https://doi.org/10.1111/j.1365-2826.2009.01879.x neurons independently of the arcuate nucleus. Psychoneuroendocrinology, 67, Benz, V., Bloch, M., Wardat, S., Böhm, C., Maurer, L., Mahmoodzadeh, S., . . . 27-39. https://doi.org/10.1016/j.psyneuen.2016.01.027 Kintscher, U. (2012). Sexual dimorphic regulation of body weight Callard, G. V., Petro, Z., & Ryan, K. J. (1978). Phylogenetic Distribution of dynamics and adipose tissue lipolysis. PLoS One, 7(5), e37794. Aromatase and Other Androgen-Converting Enzymes in the Central https://doi.org/10.1371/journal.pone.0037794 Nervous System*. Endocrinology, 103(6), 2283-2290. Bethin, K. E., Vogt, S. K., & Muglia, L. J. (2000). Interleukin-6 is an essential, https://doi.org/10.1210/endo-103-6-2283 corticotropin-releasing hormone-independent stimulator of the adrenal 45 46 Ivana Maric Ivana Maric Cannon, B., & Nedergaard, J. (2004). Brown adipose tissue: function and Cox, J. E., & Sims, J. S. (1988). Ventromedial hypothalamic and paraventricular physiological significance. Physiol Rev, 84(1), 277-359. nucleus lesions damage a common system to produce hyperphagia. https://doi.org/10.1152/physrev.00015.2003 Behavioural Brain Research, 28(3), 297-308. Carlini, V. P., Varas, M. M., Cragnolini, A. B., Schiöth, H. B., Scimonelli, T. N., https://doi.org/https://doi.org/10.1016/0166-4328(88)90132-5 & de Barioglio, S. R. (2004). Differential role of the hippocampus, Cummings, D. E. (2006). Ghrelin and the short- and long-term regulation of amygdala, and dorsal raphe nucleus in regulating feeding, memory, and appetite and body weight. Physiol Behav, 89(1), 71-84. anxiety-like behavioral responses to ghrelin. Biochemical and biophysical https://doi.org/10.1016/j.physbeh.2006.05.022 research communications, 313(3), 635-641. Cummings, D. E., Purnell, J. Q., Frayo, R. S., Schmidova, K., Wisse, B. E., & Chen, H. Y., Trumbauer, M. E., Chen, A. S., Weingarth, D. T., Adams, J. R., Weigle, D. S. (2001). A preprandial rise in plasma ghrelin levels suggests Frazier, E. G., . . . Qian, S. (2004). Orexigenic Action of Peripheral a role in meal initiation in humans. Diabetes, 50(8), 1714-1719. Ghrelin Is Mediated by Neuropeptide Y and Agouti-Related Protein. Currie, P. J., Khelemsky, R., Rigsbee, E. M., Dono, L. M., Coiro, C. D., Chapman, Endocrinology, 145(6), 2607-2612. https://doi.org/10.1210/en.2003-1596 C. D., & Hinchcliff, K. (2012). Ghrelin is an orexigenic peptide and elicits Chen, J. Y., Campos, C. A., Jarvie, B. C., & Palmiter, R. D. (2018). Parabrachial anxiety-like behaviors following administration into discrete regions of CGRP Neurons Establish and Sustain Aversive Taste Memories. Neuron, the hypothalamus. Behav Brain Res, 226(1), 96-105. 100(4), 891-899.e895. https://doi.org/10.1016/j.neuron.2018.09.032 https://doi.org/10.1016/j.bbr.2011.08.037 Chondronikola, M., Volpi, E., Børsheim, E., Porter, C., Annamalai, P., Enerbäck, Curtaz, C. J., Kiesel, L., Meybohm, P., Wöckel, A., & Burek, M. (2022). Anti- S., . . . Sidossis, L. S. (2014). Brown adipose tissue improves whole-body Hormonal Therapy in Breast Cancer and Its Effect on the Blood-Brain glucose homeostasis and insulin sensitivity in humans. Diabetes, 63(12), Barrier. Cancers (Basel), 14(20). 4089-4099. https://doi.org/10.2337/db14-0746 https://doi.org/10.3390/cancers14205132 Chondronikola, M., Volpi, E., Børsheim, E., Porter, C., Saraf, M. K., Annamalai, Curtis, A. L., Bethea, T., & Valentino, R. J. (2006). Sexually dimorphic responses P., . . . Sidossis, L. S. (2016). Brown Adipose Tissue Activation Is Linked of the brain norepinephrine system to stress and corticotropin-releasing to Distinct Systemic Effects on Lipid Metabolism in Humans. Cell factor. Neuropsychopharmacology, 31(3), 544-554. Metabolism, 23(6), 1200-1206. https://doi.org/10.1038/sj.npp.1300875 https://doi.org/https://doi.org/10.1016/j.cmet.2016.04.029 Danker-Hopfe, H., Roczen, K., & Löwenstein-Wagner, U. (1995). Regulation of Chuang, J.-C., Perello, M., Sakata, I., Osborne-Lawrence, S., Savitt, J. M., Lutter, food intake during the menstrual cycle. Anthropol Anz, 53(3), 231-238. M., & Zigman, J. M. (2011). Ghrelin mediates stress-induced food- Davis, J. F., Choi, D. L., Schurdak, J. D., Fitzgerald, M. F., Clegg, D. J., Lipton, J. reward behavior in mice. The Journal of Clinical Investigation, 121(7), 2684- W., . . . Benoit, S. C. (2011). Leptin regulates energy balance and 2692. https://doi.org/10.1172/JCI57660 motivation through action at distinct neural circuits. Biol Psychiatry, 69(7), Chuang, J. C., & Zigman, J. M. (2010). Ghrelin's Roles in Stress, Mood, and 668-674. https://doi.org/10.1016/j.biopsych.2010.08.028 Anxiety Regulation. Int J Pept, 2010. Davis, K. W., Wellman, P. J., & Clifford, P. S. (2007). Augmented cocaine https://doi.org/10.1155/2010/460549 conditioned place preference in rats pretreated with systemic ghrelin. Clayton, J. A., & Collins, F. S. (2014). Policy: NIH to balance sex in cell and animal Regulatory peptides, 140(3), 148-152. studies. Nature, 509(7500), 282-283. https://doi.org/10.1038/509282a Davis, M., & Whalen, P. J. (2001). The amygdala: vigilance and emotion. Molecular Clegg, D. J., Brown, L. M., Woods, S. C., & Benoit, S. C. (2006). Gonadal psychiatry, 6(1), 13-34. Hormones Determine Sensitivity to Central Leptin and Insulin. Diabetes, Dossat, A. M., Diaz, R., Gallo, L., Panagos, A., Kay, K., & Williams, D. L. (2013). 55(4), 978-987. https://doi.org/10.2337/diabetes.55.04.06.db05-1339 Nucleus accumbens GLP-1 receptors influence meal size and palatability. Considine, R. V., Sinha, M. K., Heiman, M. L., Kriauciunas, A., Stephens, T. W., American Journal of Physiology-Endocrinology and Metabolism, 304(12), E1314- Nyce, M. R., . . . et al. (1996). Serum immunoreactive-leptin E1320. https://doi.org/10.1152/ajpendo.00137.2013 concentrations in normal-weight and obese humans. N Engl J Med, Elmquist, J. K., Elias, C. F., & Saper, C. B. (1999). From lesions to leptin: 334(5), 292-295. https://doi.org/10.1056/nejm199602013340503 hypothalamic control of food intake and body weight. Neuron, 22(2), 221- Correa-De-Araujo, R. (2006). Serious gaps: how the lack of sex/gender-based 232. research impairs health. J Womens Health (Larchmt), 15(10), 1116-1122. Espelund, U., Hansen, T. K., Højlund, K., Beck-Nielsen, H., Clausen, J. T., https://doi.org/10.1089/jwh.2006.15.1116 Hansen, B. S., . . . Frystyk, J. (2005). Fasting Unmasks a Strong Inverse Association between Ghrelin and Cortisol in Serum: Studies in Obese 47 48 Ivana Maric Ivana Maric Cannon, B., & Nedergaard, J. (2004). Brown adipose tissue: function and Cox, J. E., & Sims, J. S. (1988). Ventromedial hypothalamic and paraventricular physiological significance. Physiol Rev, 84(1), 277-359. nucleus lesions damage a common system to produce hyperphagia. https://doi.org/10.1152/physrev.00015.2003 Behavioural Brain Research, 28(3), 297-308. Carlini, V. P., Varas, M. M., Cragnolini, A. B., Schiöth, H. B., Scimonelli, T. N., https://doi.org/https://doi.org/10.1016/0166-4328(88)90132-5 & de Barioglio, S. R. (2004). Differential role of the hippocampus, Cummings, D. E. (2006). Ghrelin and the short- and long-term regulation of amygdala, and dorsal raphe nucleus in regulating feeding, memory, and appetite and body weight. Physiol Behav, 89(1), 71-84. anxiety-like behavioral responses to ghrelin. Biochemical and biophysical https://doi.org/10.1016/j.physbeh.2006.05.022 research communications, 313(3), 635-641. Cummings, D. E., Purnell, J. Q., Frayo, R. S., Schmidova, K., Wisse, B. E., & Chen, H. Y., Trumbauer, M. E., Chen, A. S., Weingarth, D. T., Adams, J. R., Weigle, D. S. (2001). A preprandial rise in plasma ghrelin levels suggests Frazier, E. G., . . . Qian, S. (2004). Orexigenic Action of Peripheral a role in meal initiation in humans. Diabetes, 50(8), 1714-1719. Ghrelin Is Mediated by Neuropeptide Y and Agouti-Related Protein. Currie, P. J., Khelemsky, R., Rigsbee, E. M., Dono, L. M., Coiro, C. D., Chapman, Endocrinology, 145(6), 2607-2612. https://doi.org/10.1210/en.2003-1596 C. D., & Hinchcliff, K. (2012). Ghrelin is an orexigenic peptide and elicits Chen, J. Y., Campos, C. A., Jarvie, B. C., & Palmiter, R. D. (2018). Parabrachial anxiety-like behaviors following administration into discrete regions of CGRP Neurons Establish and Sustain Aversive Taste Memories. Neuron, the hypothalamus. Behav Brain Res, 226(1), 96-105. 100(4), 891-899.e895. https://doi.org/10.1016/j.neuron.2018.09.032 https://doi.org/10.1016/j.bbr.2011.08.037 Chondronikola, M., Volpi, E., Børsheim, E., Porter, C., Annamalai, P., Enerbäck, Curtaz, C. J., Kiesel, L., Meybohm, P., Wöckel, A., & Burek, M. (2022). Anti- S., . . . Sidossis, L. S. (2014). Brown adipose tissue improves whole-body Hormonal Therapy in Breast Cancer and Its Effect on the Blood-Brain glucose homeostasis and insulin sensitivity in humans. Diabetes, 63(12), Barrier. Cancers (Basel), 14(20). 4089-4099. https://doi.org/10.2337/db14-0746 https://doi.org/10.3390/cancers14205132 Chondronikola, M., Volpi, E., Børsheim, E., Porter, C., Saraf, M. K., Annamalai, Curtis, A. L., Bethea, T., & Valentino, R. J. (2006). Sexually dimorphic responses P., . . . Sidossis, L. S. (2016). Brown Adipose Tissue Activation Is Linked of the brain norepinephrine system to stress and corticotropin-releasing to Distinct Systemic Effects on Lipid Metabolism in Humans. Cell factor. Neuropsychopharmacology, 31(3), 544-554. Metabolism, 23(6), 1200-1206. https://doi.org/10.1038/sj.npp.1300875 https://doi.org/https://doi.org/10.1016/j.cmet.2016.04.029 Danker-Hopfe, H., Roczen, K., & Löwenstein-Wagner, U. (1995). Regulation of Chuang, J.-C., Perello, M., Sakata, I., Osborne-Lawrence, S., Savitt, J. M., Lutter, food intake during the menstrual cycle. Anthropol Anz, 53(3), 231-238. M., & Zigman, J. M. (2011). Ghrelin mediates stress-induced food- Davis, J. F., Choi, D. L., Schurdak, J. D., Fitzgerald, M. F., Clegg, D. J., Lipton, J. reward behavior in mice. The Journal of Clinical Investigation, 121(7), 2684- W., . . . Benoit, S. C. (2011). Leptin regulates energy balance and 2692. https://doi.org/10.1172/JCI57660 motivation through action at distinct neural circuits. Biol Psychiatry, 69(7), Chuang, J. C., & Zigman, J. M. (2010). Ghrelin's Roles in Stress, Mood, and 668-674. https://doi.org/10.1016/j.biopsych.2010.08.028 Anxiety Regulation. Int J Pept, 2010. Davis, K. W., Wellman, P. J., & Clifford, P. S. (2007). Augmented cocaine https://doi.org/10.1155/2010/460549 conditioned place preference in rats pretreated with systemic ghrelin. Clayton, J. A., & Collins, F. S. (2014). Policy: NIH to balance sex in cell and animal Regulatory peptides, 140(3), 148-152. studies. Nature, 509(7500), 282-283. https://doi.org/10.1038/509282a Davis, M., & Whalen, P. J. (2001). The amygdala: vigilance and emotion. Molecular Clegg, D. J., Brown, L. M., Woods, S. C., & Benoit, S. C. (2006). Gonadal psychiatry, 6(1), 13-34. Hormones Determine Sensitivity to Central Leptin and Insulin. Diabetes, Dossat, A. M., Diaz, R., Gallo, L., Panagos, A., Kay, K., & Williams, D. L. (2013). 55(4), 978-987. https://doi.org/10.2337/diabetes.55.04.06.db05-1339 Nucleus accumbens GLP-1 receptors influence meal size and palatability. Considine, R. V., Sinha, M. K., Heiman, M. L., Kriauciunas, A., Stephens, T. W., American Journal of Physiology-Endocrinology and Metabolism, 304(12), E1314- Nyce, M. R., . . . et al. (1996). Serum immunoreactive-leptin E1320. https://doi.org/10.1152/ajpendo.00137.2013 concentrations in normal-weight and obese humans. N Engl J Med, Elmquist, J. K., Elias, C. F., & Saper, C. B. (1999). From lesions to leptin: 334(5), 292-295. https://doi.org/10.1056/nejm199602013340503 hypothalamic control of food intake and body weight. Neuron, 22(2), 221- Correa-De-Araujo, R. (2006). Serious gaps: how the lack of sex/gender-based 232. research impairs health. J Womens Health (Larchmt), 15(10), 1116-1122. Espelund, U., Hansen, T. K., Højlund, K., Beck-Nielsen, H., Clausen, J. T., https://doi.org/10.1089/jwh.2006.15.1116 Hansen, B. S., . . . Frystyk, J. (2005). Fasting Unmasks a Strong Inverse Association between Ghrelin and Cortisol in Serum: Studies in Obese 47 48 Ivana Maric Ivana Maric and Normal-Weight Subjects. The Journal of Clinical Endocrinology & Endocrinol Metab, 82(2), 414-417. Metabolism, 90(2), 741-746. https://doi.org/10.1210/jc.2004-0604 https://doi.org/10.1210/jcem.82.2.3735 Essen, G. v., Lindsund, E., Cannon, B., & Nedergaard, J. (2017). Adaptive Ge, X., Yang, H., Bednarek, M. A., Galon-Tilleman, H., Chen, P., Chen, M., . . . facultative diet-induced thermogenesis in wild-type but not in UCP1- Kaplan, D. D. (2018). LEAP2 Is an Endogenous Antagonist of the ablated mice. American Journal of Physiology-Endocrinology and Metabolism, Ghrelin Receptor. Cell Metabolism, 27(2), 461-469.e466. 313(5), E515-E527. https://doi.org/10.1152/ajpendo.00097.2017 https://doi.org/https://doi.org/10.1016/j.cmet.2017.10.016 Faulconbridge, L. F., Cummings, D. E., Kaplan, J. M., & Grill, H. J. (2003). Gentry, R. T., & Wade, G. N. (1976). Androgenic control of food intake and body Hyperphagic effects of brainstem ghrelin administration. Diabetes, 52(9), weight in male rats. J Comp Physiol Psychol, 90(1), 18-25. 2260-2265. https://doi.org/10.1037/h0077264 Fernandez, C. J., Chacko, E. C., & Pappachan, J. M. (2019). Male Obesity-related Graham, D. L., Erreger, K., Galli, A., & Stanwood, G. D. (2013). GLP-1 analog Secondary Hypogonadism - Pathophysiology, Clinical Implications and attenuates cocaine reward. Molecular Psychiatry, 18(9), 961-962. Management. Eur Endocrinol, 15(2), 83-90. https://doi.org/10.1038/mp.2012.141 https://doi.org/10.17925/ee.2019.15.2.83 Grill, H. J. (1980). Production and regulation of ingestive consummatory behavior Flegal, K. M., Kruszon-Moran, D., Carroll, M. D., Fryar, C. D., & Ogden, C. L. in the chronic decerebrate rat. Brain Research Bulletin, 5, 79-87. (2016). Trends in Obesity Among Adults in the United States, 2005 to https://doi.org/https://doi.org/10.1016/0361-9230(80)90235-X 2014. Jama, 315(21), 2284-2291. Grisel, J. E., Allen, S., Nemmani, K. V., Fee, J. R., & Carliss, R. (2005). The https://doi.org/10.1001/jama.2016.6458 influence of dextromethorphan on morphine analgesia in Swiss Webster Foster, M. T., & Pagliassotti, M. J. (2012). Metabolic alterations following visceral mice is sex-specific. Pharmacol Biochem Behav, 81(1), 131-138. fat removal and expansion: Beyond anatomic location. Adipocyte, 1(4), https://doi.org/10.1016/j.pbb.2005.03.001 192-199. https://doi.org/10.4161/adip.21756 Hanssen, M. J., Hoeks, J., Brans, B., van der Lans, A. A., Schaart, G., van den Frank, A. P., de Souza Santos, R., Palmer, B. F., & Clegg, D. J. (2019). Driessche, J. J., . . . Schrauwen, P. (2015). Short-term cold acclimation Determinants of body fat distribution in humans may provide insight improves insulin sensitivity in patients with type 2 diabetes mellitus. Nat about obesity-related health risks. Journal of Lipid Research, 60(10), 1710- Med, 21(8), 863-865. https://doi.org/10.1038/nm.3891 1719. https://doi.org/https://doi.org/10.1194/jlr.R086975 Hayes, M. R., Skibicka, K. P., Leichner, T. M., Guarnieri, D. J., DiLeone, R. J., Frisch, R. E. (1990). The right weight: body fat, menarche and ovulation. Baillieres Bence, K. K., & Grill, H. J. (2010). Endogenous leptin signaling in the Clin Obstet Gynaecol, 4(3), 419-439. https://doi.org/10.1016/s0950- caudal nucleus tractus solitarius and area postrema is required for energy 3552(05)80302-5 balance regulation. Cell Metab, 11(1), 77-83. Frye, C. A., Edinger, K., & Sumida, K. (2008). Androgen administration to aged https://doi.org/10.1016/j.cmet.2009.10.009 male mice increases anti-anxiety behavior and enhances cognitive Hetherington, A. W., & Ranson, S. W. (1940). Hypothalamic lesions and adiposity performance. Neuropsychopharmacology, 33(5), 1049-1061. in the rat. The Anatomical Record, 78(2), 149-172. https://doi.org/10.1038/sj.npp.1301498 https://doi.org/https://doi.org/10.1002/ar.1090780203 Fulton, S., Pissios, P., Manchon, Ramon P., Stiles, L., Frank, L., Pothos, E. N., . . Hill, J. W. (2012). PVN pathways controlling energy homeostasis. Indian J . Flier, J. S. (2006). Leptin Regulation of the Mesoaccumbens Dopamine Endocrinol Metab, 16(Suppl 3), S627-636. https://doi.org/10.4103/2230- Pathway. Neuron, 51(6), 811-822. 8210.105581 https://doi.org/10.1016/j.neuron.2006.09.006 Hofman, M. A., & Swaab, D. F. (1989). The sexually dimorphic nucleus of the Galer, B. S., Lee, D., Ma, T., Nagle, B., & Schlagheck, T. G. (2005). MorphiDex preoptic area in the human brain: a comparative morphometric study. J (morphine sulfate/dextromethorphan hydrobromide combination) in Anat, 164, 55-72. the treatment of chronic pain: three multicenter, randomized, double- Holt, M. K., Richards, J. E., Cook, D. R., Brierley, D. I., Williams, D. L., Reimann, blind, controlled clinical trials fail to demonstrate enhanced opioid F., . . . Trapp, S. (2019). Preproglucagon Neurons in the Nucleus of the analgesia or reduction in tolerance. Pain, 115(3), 284-295. Solitary Tract Are the Main Source of Brain GLP-1, Mediate Stress- https://doi.org/10.1016/j.pain.2005.03.004 Induced Hypophagia, and Limit Unusually Large Intakes of Food. Gambacciani, M., Ciaponi, M., Cappagli, B., Piaggesi, L., De Simone, L., Orlandi, Diabetes, 68(1), 21-33. https://doi.org/10.2337/db18-0729 R., & Genazzani, A. R. (1997). Body weight, body fat distribution, and Huo, L., Maeng, L., Bjørbæk, C., & Grill, H. J. (2007). Leptin and the Control of hormonal replacement therapy in early postmenopausal women. J Clin Food Intake: Neurons in the Nucleus of the Solitary Tract Are Activated 49 50 Ivana Maric Ivana Maric and Normal-Weight Subjects. The Journal of Clinical Endocrinology & Endocrinol Metab, 82(2), 414-417. Metabolism, 90(2), 741-746. https://doi.org/10.1210/jc.2004-0604 https://doi.org/10.1210/jcem.82.2.3735 Essen, G. v., Lindsund, E., Cannon, B., & Nedergaard, J. (2017). Adaptive Ge, X., Yang, H., Bednarek, M. A., Galon-Tilleman, H., Chen, P., Chen, M., . . . facultative diet-induced thermogenesis in wild-type but not in UCP1- Kaplan, D. D. (2018). LEAP2 Is an Endogenous Antagonist of the ablated mice. American Journal of Physiology-Endocrinology and Metabolism, Ghrelin Receptor. Cell Metabolism, 27(2), 461-469.e466. 313(5), E515-E527. https://doi.org/10.1152/ajpendo.00097.2017 https://doi.org/https://doi.org/10.1016/j.cmet.2017.10.016 Faulconbridge, L. F., Cummings, D. E., Kaplan, J. M., & Grill, H. J. (2003). Gentry, R. T., & Wade, G. N. (1976). Androgenic control of food intake and body Hyperphagic effects of brainstem ghrelin administration. Diabetes, 52(9), weight in male rats. J Comp Physiol Psychol, 90(1), 18-25. 2260-2265. https://doi.org/10.1037/h0077264 Fernandez, C. J., Chacko, E. C., & Pappachan, J. M. (2019). Male Obesity-related Graham, D. L., Erreger, K., Galli, A., & Stanwood, G. D. (2013). GLP-1 analog Secondary Hypogonadism - Pathophysiology, Clinical Implications and attenuates cocaine reward. Molecular Psychiatry, 18(9), 961-962. Management. Eur Endocrinol, 15(2), 83-90. https://doi.org/10.1038/mp.2012.141 https://doi.org/10.17925/ee.2019.15.2.83 Grill, H. J. (1980). Production and regulation of ingestive consummatory behavior Flegal, K. M., Kruszon-Moran, D., Carroll, M. D., Fryar, C. D., & Ogden, C. L. in the chronic decerebrate rat. Brain Research Bulletin, 5, 79-87. (2016). Trends in Obesity Among Adults in the United States, 2005 to https://doi.org/https://doi.org/10.1016/0361-9230(80)90235-X 2014. Jama, 315(21), 2284-2291. Grisel, J. E., Allen, S., Nemmani, K. V., Fee, J. R., & Carliss, R. (2005). The https://doi.org/10.1001/jama.2016.6458 influence of dextromethorphan on morphine analgesia in Swiss Webster Foster, M. T., & Pagliassotti, M. J. (2012). Metabolic alterations following visceral mice is sex-specific. Pharmacol Biochem Behav, 81(1), 131-138. fat removal and expansion: Beyond anatomic location. Adipocyte, 1(4), https://doi.org/10.1016/j.pbb.2005.03.001 192-199. https://doi.org/10.4161/adip.21756 Hanssen, M. J., Hoeks, J., Brans, B., van der Lans, A. A., Schaart, G., van den Frank, A. P., de Souza Santos, R., Palmer, B. F., & Clegg, D. J. (2019). Driessche, J. J., . . . Schrauwen, P. (2015). Short-term cold acclimation Determinants of body fat distribution in humans may provide insight improves insulin sensitivity in patients with type 2 diabetes mellitus. Nat about obesity-related health risks. Journal of Lipid Research, 60(10), 1710- Med, 21(8), 863-865. https://doi.org/10.1038/nm.3891 1719. https://doi.org/https://doi.org/10.1194/jlr.R086975 Hayes, M. R., Skibicka, K. P., Leichner, T. M., Guarnieri, D. J., DiLeone, R. J., Frisch, R. E. (1990). The right weight: body fat, menarche and ovulation. Baillieres Bence, K. K., & Grill, H. J. (2010). Endogenous leptin signaling in the Clin Obstet Gynaecol, 4(3), 419-439. https://doi.org/10.1016/s0950- caudal nucleus tractus solitarius and area postrema is required for energy 3552(05)80302-5 balance regulation. Cell Metab, 11(1), 77-83. Frye, C. A., Edinger, K., & Sumida, K. (2008). Androgen administration to aged https://doi.org/10.1016/j.cmet.2009.10.009 male mice increases anti-anxiety behavior and enhances cognitive Hetherington, A. W., & Ranson, S. W. (1940). Hypothalamic lesions and adiposity performance. Neuropsychopharmacology, 33(5), 1049-1061. in the rat. The Anatomical Record, 78(2), 149-172. https://doi.org/10.1038/sj.npp.1301498 https://doi.org/https://doi.org/10.1002/ar.1090780203 Fulton, S., Pissios, P., Manchon, Ramon P., Stiles, L., Frank, L., Pothos, E. N., . . Hill, J. W. (2012). PVN pathways controlling energy homeostasis. Indian J . Flier, J. S. (2006). Leptin Regulation of the Mesoaccumbens Dopamine Endocrinol Metab, 16(Suppl 3), S627-636. https://doi.org/10.4103/2230- Pathway. Neuron, 51(6), 811-822. 8210.105581 https://doi.org/10.1016/j.neuron.2006.09.006 Hofman, M. A., & Swaab, D. F. (1989). The sexually dimorphic nucleus of the Galer, B. S., Lee, D., Ma, T., Nagle, B., & Schlagheck, T. G. (2005). MorphiDex preoptic area in the human brain: a comparative morphometric study. J (morphine sulfate/dextromethorphan hydrobromide combination) in Anat, 164, 55-72. the treatment of chronic pain: three multicenter, randomized, double- Holt, M. K., Richards, J. E., Cook, D. R., Brierley, D. I., Williams, D. L., Reimann, blind, controlled clinical trials fail to demonstrate enhanced opioid F., . . . Trapp, S. (2019). Preproglucagon Neurons in the Nucleus of the analgesia or reduction in tolerance. Pain, 115(3), 284-295. Solitary Tract Are the Main Source of Brain GLP-1, Mediate Stress- https://doi.org/10.1016/j.pain.2005.03.004 Induced Hypophagia, and Limit Unusually Large Intakes of Food. Gambacciani, M., Ciaponi, M., Cappagli, B., Piaggesi, L., De Simone, L., Orlandi, Diabetes, 68(1), 21-33. https://doi.org/10.2337/db18-0729 R., & Genazzani, A. R. (1997). Body weight, body fat distribution, and Huo, L., Maeng, L., Bjørbæk, C., & Grill, H. J. (2007). Leptin and the Control of hormonal replacement therapy in early postmenopausal women. J Clin Food Intake: Neurons in the Nucleus of the Solitary Tract Are Activated 49 50 Ivana Maric Ivana Maric by Both Gastric Distension and Leptin. Endocrinology, 148(5), 2189-2197. Klausen, M. K., Thomsen, M., Wortwein, G., & Fink-Jensen, A. (2022). The role https://doi.org/10.1210/en.2006-1572 of glucagon-like peptide 1 (GLP-1) in addictive disorders. Br J Pharmacol, Institute of Medicine Forum on, N., & Nervous System, D. (2011). The National 179(4), 625-641. https://doi.org/10.1111/bph.15677 Academies Collection: Reports funded by National Institutes of Health. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., & Kangawa, K. In Sex Differences and Implications for Translational Neuroscience Research: (1999). Ghrelin is a growth-hormone-releasing acylated peptide from Workshop Summary. National Academies Press (US) stomach. Nature, 402(6762), 656-660. Copyright © 2011, National Academy of Sciences. Koob, G. F. (1992). Drugs of abuse: anatomy, pharmacology and function of https://doi.org/10.17226/13004 reward pathways. Trends in Pharmacological Sciences, 13, 177-184. Jen-Hui, T., Syun-Ruei, L., Chia-Ying, C., Yi-Jie, Y., Fong-Yi, G., Shih-Ying, N., https://doi.org/https://doi.org/10.1016/0165-6147(92)90060-J & Hau-Jie, Y. (2023). Negative emotions recruit the parabrachial nucleus Koob, G. F. (1999). Corticotropin-releasing factor, norepinephrine, and stress. efferent to the VTA to disengage instrumental food seeking. The Journal Biol Psychiatry, 46(9), 1167-1180. https://doi.org/10.1016/s0006- of Neuroscience, JN-RM-2114-2122. 3223(99)00164-x https://doi.org/10.1523/JNEUROSCI.2114-22.2023 Krude, H., Biebermann, H., Luck, W., Horn, R., Brabant, G., & Grüters, A. Jerlhag, E., Egecioglu, E., Dickson, S. L., & Engel, J. A. (2010). Ghrelin receptor (1998). Severe early-onset obesity, adrenal insufficiency and red hair antagonism attenuates cocaine- and amphetamine-induced locomotor pigmentation caused by POMC mutations in humans. Nat Genet, 19(2), stimulation, accumbal dopamine release, and conditioned place 155-157. https://doi.org/10.1038/509 preference. Psychopharmacology (Berl), 211(4), 415-422. Kulterer, O. C., Herz, C. T., Prager, M., Schmöltzer, C., Langer, F. B., Prager, G., https://doi.org/10.1007/s00213-010-1907-7 . . . Kiefer, F. W. (2022). Brown Adipose Tissue Prevalence Is Lower in Jerlhag, E., Egecioglu, E., Landgren, S., Salomé, N., Heilig, M., Moechars, D., . . Obesity but Its Metabolic Activity Is Intact. Front Endocrinol (Lausanne), . Engel, J. A. (2009). Requirement of central ghrelin signaling for alcohol 13, 858417. https://doi.org/10.3389/fendo.2022.858417 reward. Proc Natl Acad Sci U S A, 106(27), 11318-11323. la Fleur, S. E., Luijendijk, M. C., van Rozen, A. J., Kalsbeek, A., & Adan, R. A. https://doi.org/10.1073/pnas.0812809106 (2011). A free-choice high-fat high-sugar diet induces glucose intolerance Jerlhag, E., & Engel, J. A. (2011). Ghrelin receptor antagonism attenuates and insulin unresponsiveness to a glucose load not explained by obesity. nicotine-induced locomotor stimulation, accumbal dopamine release and Int J Obes (Lond), 35(4), 595-604. https://doi.org/10.1038/ijo.2010.164 conditioned place preference in mice. Drug Alcohol Depend, 117(2-3), 126- la Fleur, S. E., Luijendijk, M. C. M., van der Zwaal, E. M., Brans, M. A. D., & 131. https://doi.org/10.1016/j.drugalcdep.2011.01.010 Adan, R. A. H. (2014). The snacking rat as model of human obesity: Kaplan, J. M., Seeley, R. J., & Grill, H. J. (1993). Daily caloric intake in intact and effects of a free-choice high-fat high-sugar diet on meal patterns. chronic decerebrate rats. Behav Neurosci, 107(5), 876-881. International Journal of Obesity, 38(5), 643-649. Kaufman, J. M., & Vermeulen, A. (2005). The Decline of Androgen Levels in https://doi.org/10.1038/ijo.2013.159 Elderly Men and Its Clinical and Therapeutic Implications. Endocrine Le May, M. V., Hume, C., Sabatier, N., Schéle, E., Bake, T., Bergström, U., . . . Reviews, 26(6), 833-876. https://doi.org/10.1210/er.2004-0013 Dickson, S. L. (2019). Activation of the rat hypothalamic Khaodhiar, L., Ling, P. R., Blackburn, G. L., & Bistrian, B. R. (2004). Serum levels supramammillary nucleus by food anticipation, food restriction or ghrelin of interleukin-6 and C-reactive protein correlate with body mass index administration. Journal of Neuroendocrinology, 31(7), e12676. across the broad range of obesity. JPEN J Parenter Enteral Nutr, 28(6), https://doi.org/https://doi.org/10.1111/jne.12676 410-415. https://doi.org/10.1177/0148607104028006410 Liu, K. A., & Mager, N. A. (2016). Women's involvement in clinical trials: Kim, N. R., David, K., Corbeels, K., Khalil, R., Antonio, L., Schollaert, D., . . . historical perspective and future implications. Pharm Pract (Granada), Dubois, V. (2021). Testosterone Reduces Body Fat in Male Mice by 14(1), 708. https://doi.org/10.18549/PharmPract.2016.01.708 Stimulation of Physical Activity Via Extrahypothalamic ERα Signaling. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data Endocrinology, 162(6). https://doi.org/10.1210/endocr/bqab045 using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Kissileff, H. R., Pi-Sunyer, F. X., Thornton, J., & Smith, G. P. (1981). C-terminal Methods, 25(4), 402-408. https://doi.org/10.1006/meth.2001.1262 octapeptide of cholecystokinin decreases food intake in man. The Lockie, S. H., Dinan, T., Lawrence, A. J., Spencer, S. J., & Andrews, Z. B. (2015). American journal of clinical nutrition, 34(2), 154-160. Diet-induced obesity causes ghrelin resistance in reward processing tasks. Psychoneuroendocrinology, 62, 114-120. https://doi.org/https://doi.org/10.1016/j.psyneuen.2015.08.004 51 52 Ivana Maric Ivana Maric by Both Gastric Distension and Leptin. Endocrinology, 148(5), 2189-2197. Klausen, M. K., Thomsen, M., Wortwein, G., & Fink-Jensen, A. (2022). The role https://doi.org/10.1210/en.2006-1572 of glucagon-like peptide 1 (GLP-1) in addictive disorders. Br J Pharmacol, Institute of Medicine Forum on, N., & Nervous System, D. (2011). The National 179(4), 625-641. https://doi.org/10.1111/bph.15677 Academies Collection: Reports funded by National Institutes of Health. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., & Kangawa, K. In Sex Differences and Implications for Translational Neuroscience Research: (1999). Ghrelin is a growth-hormone-releasing acylated peptide from Workshop Summary. National Academies Press (US) stomach. Nature, 402(6762), 656-660. Copyright © 2011, National Academy of Sciences. Koob, G. F. (1992). Drugs of abuse: anatomy, pharmacology and function of https://doi.org/10.17226/13004 reward pathways. Trends in Pharmacological Sciences, 13, 177-184. Jen-Hui, T., Syun-Ruei, L., Chia-Ying, C., Yi-Jie, Y., Fong-Yi, G., Shih-Ying, N., https://doi.org/https://doi.org/10.1016/0165-6147(92)90060-J & Hau-Jie, Y. (2023). Negative emotions recruit the parabrachial nucleus Koob, G. F. (1999). Corticotropin-releasing factor, norepinephrine, and stress. efferent to the VTA to disengage instrumental food seeking. The Journal Biol Psychiatry, 46(9), 1167-1180. https://doi.org/10.1016/s0006- of Neuroscience, JN-RM-2114-2122. 3223(99)00164-x https://doi.org/10.1523/JNEUROSCI.2114-22.2023 Krude, H., Biebermann, H., Luck, W., Horn, R., Brabant, G., & Grüters, A. Jerlhag, E., Egecioglu, E., Dickson, S. L., & Engel, J. A. (2010). Ghrelin receptor (1998). Severe early-onset obesity, adrenal insufficiency and red hair antagonism attenuates cocaine- and amphetamine-induced locomotor pigmentation caused by POMC mutations in humans. Nat Genet, 19(2), stimulation, accumbal dopamine release, and conditioned place 155-157. https://doi.org/10.1038/509 preference. Psychopharmacology (Berl), 211(4), 415-422. Kulterer, O. C., Herz, C. T., Prager, M., Schmöltzer, C., Langer, F. B., Prager, G., https://doi.org/10.1007/s00213-010-1907-7 . . . Kiefer, F. W. (2022). Brown Adipose Tissue Prevalence Is Lower in Jerlhag, E., Egecioglu, E., Landgren, S., Salomé, N., Heilig, M., Moechars, D., . . Obesity but Its Metabolic Activity Is Intact. Front Endocrinol (Lausanne), . Engel, J. A. (2009). Requirement of central ghrelin signaling for alcohol 13, 858417. https://doi.org/10.3389/fendo.2022.858417 reward. Proc Natl Acad Sci U S A, 106(27), 11318-11323. la Fleur, S. E., Luijendijk, M. C., van Rozen, A. J., Kalsbeek, A., & Adan, R. A. https://doi.org/10.1073/pnas.0812809106 (2011). A free-choice high-fat high-sugar diet induces glucose intolerance Jerlhag, E., & Engel, J. A. (2011). Ghrelin receptor antagonism attenuates and insulin unresponsiveness to a glucose load not explained by obesity. nicotine-induced locomotor stimulation, accumbal dopamine release and Int J Obes (Lond), 35(4), 595-604. https://doi.org/10.1038/ijo.2010.164 conditioned place preference in mice. Drug Alcohol Depend, 117(2-3), 126- la Fleur, S. E., Luijendijk, M. C. M., van der Zwaal, E. M., Brans, M. A. D., & 131. https://doi.org/10.1016/j.drugalcdep.2011.01.010 Adan, R. A. H. (2014). The snacking rat as model of human obesity: Kaplan, J. M., Seeley, R. J., & Grill, H. J. (1993). Daily caloric intake in intact and effects of a free-choice high-fat high-sugar diet on meal patterns. chronic decerebrate rats. Behav Neurosci, 107(5), 876-881. International Journal of Obesity, 38(5), 643-649. Kaufman, J. M., & Vermeulen, A. (2005). The Decline of Androgen Levels in https://doi.org/10.1038/ijo.2013.159 Elderly Men and Its Clinical and Therapeutic Implications. Endocrine Le May, M. V., Hume, C., Sabatier, N., Schéle, E., Bake, T., Bergström, U., . . . Reviews, 26(6), 833-876. https://doi.org/10.1210/er.2004-0013 Dickson, S. L. (2019). Activation of the rat hypothalamic Khaodhiar, L., Ling, P. R., Blackburn, G. L., & Bistrian, B. R. (2004). Serum levels supramammillary nucleus by food anticipation, food restriction or ghrelin of interleukin-6 and C-reactive protein correlate with body mass index administration. Journal of Neuroendocrinology, 31(7), e12676. across the broad range of obesity. JPEN J Parenter Enteral Nutr, 28(6), https://doi.org/https://doi.org/10.1111/jne.12676 410-415. https://doi.org/10.1177/0148607104028006410 Liu, K. A., & Mager, N. A. (2016). Women's involvement in clinical trials: Kim, N. R., David, K., Corbeels, K., Khalil, R., Antonio, L., Schollaert, D., . . . historical perspective and future implications. Pharm Pract (Granada), Dubois, V. (2021). Testosterone Reduces Body Fat in Male Mice by 14(1), 708. https://doi.org/10.18549/PharmPract.2016.01.708 Stimulation of Physical Activity Via Extrahypothalamic ERα Signaling. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data Endocrinology, 162(6). https://doi.org/10.1210/endocr/bqab045 using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Kissileff, H. R., Pi-Sunyer, F. X., Thornton, J., & Smith, G. P. (1981). C-terminal Methods, 25(4), 402-408. https://doi.org/10.1006/meth.2001.1262 octapeptide of cholecystokinin decreases food intake in man. The Lockie, S. H., Dinan, T., Lawrence, A. J., Spencer, S. J., & Andrews, Z. B. (2015). American journal of clinical nutrition, 34(2), 154-160. Diet-induced obesity causes ghrelin resistance in reward processing tasks. Psychoneuroendocrinology, 62, 114-120. https://doi.org/https://doi.org/10.1016/j.psyneuen.2015.08.004 51 52 Ivana Maric Ivana Maric Lutter, M., Sakata, I., Osborne-Lawrence, S., Rovinsky, S. A., Anderson, J. G., mechanisms. Molecular Psychiatry, 24(1), 18-33. Jung, S., . . . Nestler, E. J. (2008). The orexigenic hormone ghrelin https://doi.org/10.1038/s41380-018-0017-5 defends against depressive symptoms of chronic stress. Nature neuroscience, Miller, W. L., & Auchus, R. J. (2011). The molecular biology, biochemistry, and 11(7), 752-753. physiology of human steroidogenesis and its disorders. Endocr Rev, 32(1), López-Ferreras, L., Longo, F., Richard, J. E., Eerola, K., Shevchouk, O. T., 81-151. https://doi.org/10.1210/er.2010-0013 Tuzinovic, M., & Skibicka, K. P. (2021). Key role for hypothalamic Miyahara, S., Komori, T., Fujiwara, R., Shizuya, K., Yamamoto, M., Ohmori, M., interleukin-6 in food-motivated behavior and body weight regulation. & Okazaki, Y. (2000). Effects of repeated stress on expression of Psychoneuroendocrinology, 131, 105284. interleukin-6 (IL-6) and IL-6 receptor mRNAs in rat hypothalamus and https://doi.org/https://doi.org/10.1016/j.psyneuen.2021.105284 midbrain. Life Sci, 66(6), Pl93-98. https://doi.org/10.1016/s0024- López-Ferreras, L., Richard, J. E., Anderberg, R. H., Nilsson, F. H., Olandersson, 3205(99)00626-8 K., Kanoski, S. E., & Skibicka, K. P. (2017). Ghrelin's control of food Mogil, J. S. (2020). Qualitative sex differences in pain processing: emerging reward and body weight in the lateral hypothalamic area is sexually evidence of a biased literature. Nature Reviews Neuroscience, 21(7), 353-365. dimorphic. Physiology & behavior, 176, 40-49. https://doi.org/10.1038/s41583-020-0310-6 M'Kadmi, C., Cabral, A., Barrile, F., Giribaldi, J., Cantel, S., Damian, M., . . . Mohamed-Ali, V., Goodrick, S., Rawesh, A., Katz, D., Miles, J., Yudkin, J., . . . Fehrentz, J. A. (2019). N-Terminal Liver-Expressed Antimicrobial Coppack, S. (1997). Subcutaneous adipose tissue releases interleukin-6, Peptide 2 (LEAP2) Region Exhibits Inverse Agonist Activity toward the but not tumor necrosis factor-α, in vivo. The Journal of Clinical Endocrinology Ghrelin Receptor. J Med Chem, 62(2), 965-973. & Metabolism, 82(12), 4196-4200. https://doi.org/10.1021/acs.jmedchem.8b01644 Montague, C. T., Farooqi, I. S., Whitehead, J. P., Soos, M. A., Rau, H., Wareham, MacLusky, N. J., & McEwen, B. S. (1980). Progestin receptors in rat brain: N. J., . . . O'Rahilly, S. (1997). Congenital leptin deficiency is associated distribution and properties of cytoplasmic progestin-binding sites. with severe early-onset obesity in humans. Nature, 387(6636), 903-908. Endocrinology, 106(1), 192-202. https://doi.org/10.1210/endo-106-1-192 https://doi.org/10.1038/43185 Margules, D. L., & Olds, J. (1962). Identical "feeding" and "rewarding" systems Monteiro, R., & Azevedo, I. (2010). Chronic inflammation in obesity and the in the lateral hypothalamus of rats. Science, 135(3501), 374-375. metabolic syndrome. Mediators Inflamm, 2010. https://doi.org/10.1126/science.135.3501.374 https://doi.org/10.1155/2010/289645 Matheny, M., Shapiro, A., Tümer, N., & Scarpace, P. J. (2011). Region-specific Morris, L. S., McCall, J. G., Charney, D. S., & Murrough, J. W. (2020). The role diet-induced and leptin-induced cellular leptin resistance includes the of the locus coeruleus in the generation of pathological anxiety. Brain ventral tegmental area in rats. Neuropharmacology, 60(2-3), 480-487. Neurosci Adv, 4, 2398212820930321. https://doi.org/10.1016/j.neuropharm.2010.11.002 https://doi.org/10.1177/2398212820930321 Mauvais-Jarvis, F., Bairey Merz, N., Barnes, P. J., Brinton, R. D., Carrero, J.-J., Morselli, E., Frank, A. P., Palmer, B. F., Rodriguez-Navas, C., Criollo, A., & DeMeo, D. L., . . . Suzuki, A. (2020). Sex and gender: modifiers of health, Clegg, D. J. (2016). A sexually dimorphic hypothalamic response to disease, and medicine. The Lancet, 396(10250), 565-582. chronic high-fat diet consumption. Int J Obes (Lond), 40(2), 206-209. https://doi.org/https://doi.org/10.1016/S0140-6736(20)31561-0 https://doi.org/10.1038/ijo.2015.114 McTernan, P. G., Anwar, A., Eggo, M. C., Barnett, A. H., Stewart, P. M., & Nedergaard, J., & Cannon, B. (2010). The Changed Metabolic World with Human Kumar, S. (2000). Gender differences in the regulation of P450 Brown Adipose Tissue: Therapeutic Visions. Cell Metabolism, 11(4), 268- aromatase expression and activity in human adipose tissue. International 272. https://doi.org/https://doi.org/10.1016/j.cmet.2010.03.007 Journal of Obesity, 24(7), 875-881. https://doi.org/10.1038/sj.ijo.0801254 Nohara, K., Zhang, Y., Waraich, R. S., Laque, A., Tiano, J. P., Tong, J., . . . Mercer, J. G., Hoggard, N., Williams, L. M., Lawrence, C. B., Hannah, L. T., Mauvais-Jarvis, F. (2011). Early-life exposure to testosterone programs Morgan, P. J., & Trayhurn, P. (1996). Coexpression of leptin receptor the hypothalamic melanocortin system. Endocrinology, 152(4), 1661-1669. and preproneuropeptide Y mRNA in arcuate nucleus of mouse https://doi.org/10.1210/en.2010-1288 hypothalamus. J Neuroendocrinol, 8(10), 733-735. Office, U. S. G. A. (2001). Drug Safety: Most Drugs Withdrawn in Recent Years https://doi.org/10.1046/j.1365-2826.1996.05161.x Had Greater Health Milaneschi, Y., Simmons, W. K., van Rossum, E. F. C., & Penninx, B. W. J. H. Risks for Women. In GAO Reports and Comptroller General Decisions. United States (2019). Depression and obesity: evidence of shared biological Government Accountability Office. 53 54 Ivana Maric Ivana Maric Lutter, M., Sakata, I., Osborne-Lawrence, S., Rovinsky, S. A., Anderson, J. G., mechanisms. Molecular Psychiatry, 24(1), 18-33. Jung, S., . . . Nestler, E. J. (2008). The orexigenic hormone ghrelin https://doi.org/10.1038/s41380-018-0017-5 defends against depressive symptoms of chronic stress. Nature neuroscience, Miller, W. L., & Auchus, R. J. (2011). The molecular biology, biochemistry, and 11(7), 752-753. physiology of human steroidogenesis and its disorders. Endocr Rev, 32(1), López-Ferreras, L., Longo, F., Richard, J. E., Eerola, K., Shevchouk, O. T., 81-151. https://doi.org/10.1210/er.2010-0013 Tuzinovic, M., & Skibicka, K. P. (2021). Key role for hypothalamic Miyahara, S., Komori, T., Fujiwara, R., Shizuya, K., Yamamoto, M., Ohmori, M., interleukin-6 in food-motivated behavior and body weight regulation. & Okazaki, Y. (2000). Effects of repeated stress on expression of Psychoneuroendocrinology, 131, 105284. interleukin-6 (IL-6) and IL-6 receptor mRNAs in rat hypothalamus and https://doi.org/https://doi.org/10.1016/j.psyneuen.2021.105284 midbrain. Life Sci, 66(6), Pl93-98. https://doi.org/10.1016/s0024- López-Ferreras, L., Richard, J. E., Anderberg, R. H., Nilsson, F. H., Olandersson, 3205(99)00626-8 K., Kanoski, S. E., & Skibicka, K. P. (2017). Ghrelin's control of food Mogil, J. S. (2020). Qualitative sex differences in pain processing: emerging reward and body weight in the lateral hypothalamic area is sexually evidence of a biased literature. Nature Reviews Neuroscience, 21(7), 353-365. dimorphic. Physiology & behavior, 176, 40-49. https://doi.org/10.1038/s41583-020-0310-6 M'Kadmi, C., Cabral, A., Barrile, F., Giribaldi, J., Cantel, S., Damian, M., . . . Mohamed-Ali, V., Goodrick, S., Rawesh, A., Katz, D., Miles, J., Yudkin, J., . . . Fehrentz, J. A. (2019). N-Terminal Liver-Expressed Antimicrobial Coppack, S. (1997). Subcutaneous adipose tissue releases interleukin-6, Peptide 2 (LEAP2) Region Exhibits Inverse Agonist Activity toward the but not tumor necrosis factor-α, in vivo. The Journal of Clinical Endocrinology Ghrelin Receptor. J Med Chem, 62(2), 965-973. & Metabolism, 82(12), 4196-4200. https://doi.org/10.1021/acs.jmedchem.8b01644 Montague, C. T., Farooqi, I. S., Whitehead, J. P., Soos, M. A., Rau, H., Wareham, MacLusky, N. J., & McEwen, B. S. (1980). Progestin receptors in rat brain: N. J., . . . O'Rahilly, S. (1997). Congenital leptin deficiency is associated distribution and properties of cytoplasmic progestin-binding sites. with severe early-onset obesity in humans. Nature, 387(6636), 903-908. Endocrinology, 106(1), 192-202. https://doi.org/10.1210/endo-106-1-192 https://doi.org/10.1038/43185 Margules, D. L., & Olds, J. (1962). Identical "feeding" and "rewarding" systems Monteiro, R., & Azevedo, I. (2010). Chronic inflammation in obesity and the in the lateral hypothalamus of rats. Science, 135(3501), 374-375. metabolic syndrome. Mediators Inflamm, 2010. https://doi.org/10.1126/science.135.3501.374 https://doi.org/10.1155/2010/289645 Matheny, M., Shapiro, A., Tümer, N., & Scarpace, P. J. (2011). Region-specific Morris, L. S., McCall, J. G., Charney, D. S., & Murrough, J. W. (2020). The role diet-induced and leptin-induced cellular leptin resistance includes the of the locus coeruleus in the generation of pathological anxiety. Brain ventral tegmental area in rats. Neuropharmacology, 60(2-3), 480-487. Neurosci Adv, 4, 2398212820930321. https://doi.org/10.1016/j.neuropharm.2010.11.002 https://doi.org/10.1177/2398212820930321 Mauvais-Jarvis, F., Bairey Merz, N., Barnes, P. J., Brinton, R. D., Carrero, J.-J., Morselli, E., Frank, A. P., Palmer, B. F., Rodriguez-Navas, C., Criollo, A., & DeMeo, D. L., . . . Suzuki, A. (2020). Sex and gender: modifiers of health, Clegg, D. J. (2016). A sexually dimorphic hypothalamic response to disease, and medicine. The Lancet, 396(10250), 565-582. chronic high-fat diet consumption. Int J Obes (Lond), 40(2), 206-209. https://doi.org/https://doi.org/10.1016/S0140-6736(20)31561-0 https://doi.org/10.1038/ijo.2015.114 McTernan, P. G., Anwar, A., Eggo, M. C., Barnett, A. H., Stewart, P. M., & Nedergaard, J., & Cannon, B. (2010). The Changed Metabolic World with Human Kumar, S. (2000). Gender differences in the regulation of P450 Brown Adipose Tissue: Therapeutic Visions. Cell Metabolism, 11(4), 268- aromatase expression and activity in human adipose tissue. International 272. https://doi.org/https://doi.org/10.1016/j.cmet.2010.03.007 Journal of Obesity, 24(7), 875-881. https://doi.org/10.1038/sj.ijo.0801254 Nohara, K., Zhang, Y., Waraich, R. S., Laque, A., Tiano, J. P., Tong, J., . . . Mercer, J. G., Hoggard, N., Williams, L. M., Lawrence, C. B., Hannah, L. T., Mauvais-Jarvis, F. (2011). Early-life exposure to testosterone programs Morgan, P. J., & Trayhurn, P. (1996). Coexpression of leptin receptor the hypothalamic melanocortin system. Endocrinology, 152(4), 1661-1669. and preproneuropeptide Y mRNA in arcuate nucleus of mouse https://doi.org/10.1210/en.2010-1288 hypothalamus. J Neuroendocrinol, 8(10), 733-735. Office, U. S. G. A. (2001). Drug Safety: Most Drugs Withdrawn in Recent Years https://doi.org/10.1046/j.1365-2826.1996.05161.x Had Greater Health Milaneschi, Y., Simmons, W. K., van Rossum, E. F. C., & Penninx, B. W. J. H. Risks for Women. In GAO Reports and Comptroller General Decisions. United States (2019). Depression and obesity: evidence of shared biological Government Accountability Office. 53 54 Ivana Maric Ivana Maric Palmer, B. F., & Clegg, D. J. (2015). The sexual dimorphism of obesity. Mol Cell Rocks, D., Cham, H., & Kundakovic, M. (2022). Why the estrous cycle matters Endocrinol, 402, 113-119. https://doi.org/10.1016/j.mce.2014.11.029 for neuroscience. Biology of Sex Differences, 13(1), 62. Palmer, K., & Gray, J. M. (1986). Central vs. peripheral effects of estrogen on https://doi.org/10.1186/s13293-022-00466-8 food intake and lipoprotein lipase activity in ovariectomized rats. Physiol Rodriguez-Cuenca, S., Pujol, E., Justo, R., Frontera, M., Oliver, J., Gianotti, M., Behav, 37(1), 187-189. https://doi.org/10.1016/0031-9384(86)90404-x & Roca, P. (2002). Sex-dependent thermogenesis, differences in Palmiter, R. D. (2007). Is dopamine a physiologically relevant mediator of feeding mitochondrial morphology and function, and adrenergic response in behavior? Trends in Neurosciences, 30(8), 375-381. brown adipose tissue. J Biol Chem, 277(45), 42958-42963. https://doi.org/10.1016/j.tins.2007.06.004 https://doi.org/10.1074/jbc.M207229200 Patterson, Z. R., Khazall, R., Mackay, H., Anisman, H., & Abizaid, A. (2013). Roney, J. R., & Simmons, Z. L. (2017). Ovarian hormone fluctuations predict Central ghrelin signaling mediates the metabolic response of C57BL/6 within-cycle shifts in women's food intake. Horm Behav, 90, 8-14. male mice to chronic social defeat stress. Endocrinology, 154(3), 1080-1091. https://doi.org/10.1016/j.yhbeh.2017.01.009 https://doi.org/10.1210/en.2012-1834 Roselli, C. E., Abdelgadir, S. E., Rønnekleiv, O. K., & Klosterman, S. A. (1998). Perello, M., Cabral, A., Cornejo, M. P., De Francesco, P. N., Fernandez, G., & Anatomic Distribution and Regulation of Aromatase Gene Expression Uriarte, M. (2019). Brain accessibility delineates the central effects of in the Rat Brain1. Biology of Reproduction, 58(1), 79-87. circulating ghrelin. Journal of Neuroendocrinology, 31(7), e12677. https://doi.org/10.1095/biolreprod58.1.79 https://doi.org/https://doi.org/10.1111/jne.12677 Rosenbaum, M., Nicolson, M., Hirsch, J., Heymsfield, S. B., Gallagher, D., Chu, Pfannenberg, C., Werner, M. K., Ripkens, S., Stef, I., Deckert, A., Schmadl, M., . F., & Leibel, R. L. (1996). Effects of gender, body composition, and . . Stefan, N. (2010). Impact of age on the relationships of brown adipose menopause on plasma concentrations of leptin. J Clin Endocrinol Metab, tissue with sex and adiposity in humans. Diabetes, 59(7), 1789-1793. 81(9), 3424-3427. https://doi.org/10.1210/jcem.81.9.8784109 https://doi.org/10.2337/db10-0004 Santollo, J., & Eckel, L. A. (2008). The orexigenic effect of melanin-concentrating Prendergast, B. J., Onishi, K. G., & Zucker, I. (2014). Female mice liberated for hormone (MCH) is influenced by sex and stage of the estrous cycle. inclusion in neuroscience and biomedical research. Neurosci Biobehav Rev, Physiol Behav, 93(4-5), 842-850. 40, 1-5. https://doi.org/10.1016/j.neubiorev.2014.01.001 https://doi.org/10.1016/j.physbeh.2007.11.050 Qu, D., Ludwig, D. S., Gammeltoft, S., Piper, M., Pelleymounter, M. A., Cullen, Sciolino, N. R., Hsiang, M., Mazzone, C. M., Wilson, L. R., Plummer, N. W., M. J., . . . Maratos-Flier, E. (1996). A role for melanin-concentrating Amin, J., . . . Jensen, P. (2022). Natural locus coeruleus dynamics during hormone in the central regulation of feeding behaviour. Nature, feeding. Sci Adv, 8(33), eabn9134. 380(6571), 243-247. https://doi.org/10.1038/380243a0 https://doi.org/10.1126/sciadv.abn9134 Rand, K. L., Otte, J. L., Flockhart, D., Hayes, D., Storniolo, A. M., Stearns, V., . . Sclafani, A., & Springer, D. (1976). Dietary obesity in adult rats: similarities to . Carpenter, J. S. (2011). Modeling hot flushes and quality of life in breast hypothalamic and human obesity syndromes. Physiol Behav, 17(3), 461- cancer survivors. Climacteric, 14(1), 171-180. 471. https://doi.org/10.1016/0031-9384(76)90109-8 https://doi.org/10.3109/13697131003717070 Seeley, R. J., van Dijk, G., Campfield, L. A., Smith, F. J., Burn, P., Nelligan, J. A., Richard, J. E., López-Ferreras, L., Anderberg, R. H., Olandersson, K., & Skibicka, . . . Schwartz, M. W. (1996). Intraventricular leptin reduces food intake K. P. (2017). Estradiol is a critical regulator of food-reward behavior. and body weight of lean rats but not obese Zucker rats. Horm Metab Res, Psychoneuroendocrinology, 78, 193-202. 28(12), 664-668. https://doi.org/10.1055/s-2007-979874 https://doi.org/10.1016/j.psyneuen.2017.01.014 Seeley, R. J., Yagaloff, K. A., Fisher, S. L., Burn, P., Thiele, T. E., van Dijk, G., . . Rinaman, L. (2010). Ascending projections from the caudal visceral nucleus of . Schwartz, M. W. (1997). Melanocortin receptors in leptin effects. Nature, the solitary tract to brain regions involved in food intake and energy 390(6658), 349. https://doi.org/10.1038/37016 expenditure. Brain Research, 1350, 18-34. Shi, H., Strader, A. D., Woods, S. C., & Seeley, R. J. (2007). Sexually dimorphic https://doi.org/10.1016/j.brainres.2010.03.059 responses to fat loss after caloric restriction or surgical lipectomy. Am J Rivera, H. M., & Eckel, L. A. (2010). Activation of Central, But Not Peripheral, Physiol Endocrinol Metab, 293(1), E316-326. Estrogen Receptors Is Necessary for Estradiol’s Anorexigenic Effect in https://doi.org/10.1152/ajpendo.00710.2006 Ovariectomized Rats. Endocrinology, 151(12), 5680-5688. Shughrue, P. J., Lane, M. V., & Merchenthaler, I. (1997). Comparative distribution https://doi.org/10.1210/en.2010-0731 of estrogen receptor-α and -β mRNA in the rat central nervous system. Journal of Comparative Neurology, 388(4), 507-525. 55 56 Ivana Maric Ivana Maric Palmer, B. F., & Clegg, D. J. (2015). The sexual dimorphism of obesity. Mol Cell Rocks, D., Cham, H., & Kundakovic, M. (2022). Why the estrous cycle matters Endocrinol, 402, 113-119. https://doi.org/10.1016/j.mce.2014.11.029 for neuroscience. Biology of Sex Differences, 13(1), 62. Palmer, K., & Gray, J. M. (1986). Central vs. peripheral effects of estrogen on https://doi.org/10.1186/s13293-022-00466-8 food intake and lipoprotein lipase activity in ovariectomized rats. Physiol Rodriguez-Cuenca, S., Pujol, E., Justo, R., Frontera, M., Oliver, J., Gianotti, M., Behav, 37(1), 187-189. https://doi.org/10.1016/0031-9384(86)90404-x & Roca, P. (2002). Sex-dependent thermogenesis, differences in Palmiter, R. D. (2007). Is dopamine a physiologically relevant mediator of feeding mitochondrial morphology and function, and adrenergic response in behavior? Trends in Neurosciences, 30(8), 375-381. brown adipose tissue. J Biol Chem, 277(45), 42958-42963. https://doi.org/10.1016/j.tins.2007.06.004 https://doi.org/10.1074/jbc.M207229200 Patterson, Z. R., Khazall, R., Mackay, H., Anisman, H., & Abizaid, A. (2013). Roney, J. R., & Simmons, Z. L. (2017). Ovarian hormone fluctuations predict Central ghrelin signaling mediates the metabolic response of C57BL/6 within-cycle shifts in women's food intake. Horm Behav, 90, 8-14. male mice to chronic social defeat stress. Endocrinology, 154(3), 1080-1091. https://doi.org/10.1016/j.yhbeh.2017.01.009 https://doi.org/10.1210/en.2012-1834 Roselli, C. E., Abdelgadir, S. E., Rønnekleiv, O. K., & Klosterman, S. A. (1998). Perello, M., Cabral, A., Cornejo, M. P., De Francesco, P. N., Fernandez, G., & Anatomic Distribution and Regulation of Aromatase Gene Expression Uriarte, M. (2019). Brain accessibility delineates the central effects of in the Rat Brain1. Biology of Reproduction, 58(1), 79-87. circulating ghrelin. Journal of Neuroendocrinology, 31(7), e12677. https://doi.org/10.1095/biolreprod58.1.79 https://doi.org/https://doi.org/10.1111/jne.12677 Rosenbaum, M., Nicolson, M., Hirsch, J., Heymsfield, S. B., Gallagher, D., Chu, Pfannenberg, C., Werner, M. K., Ripkens, S., Stef, I., Deckert, A., Schmadl, M., . F., & Leibel, R. L. (1996). Effects of gender, body composition, and . . Stefan, N. (2010). Impact of age on the relationships of brown adipose menopause on plasma concentrations of leptin. J Clin Endocrinol Metab, tissue with sex and adiposity in humans. Diabetes, 59(7), 1789-1793. 81(9), 3424-3427. https://doi.org/10.1210/jcem.81.9.8784109 https://doi.org/10.2337/db10-0004 Santollo, J., & Eckel, L. A. (2008). The orexigenic effect of melanin-concentrating Prendergast, B. J., Onishi, K. G., & Zucker, I. (2014). Female mice liberated for hormone (MCH) is influenced by sex and stage of the estrous cycle. inclusion in neuroscience and biomedical research. Neurosci Biobehav Rev, Physiol Behav, 93(4-5), 842-850. 40, 1-5. https://doi.org/10.1016/j.neubiorev.2014.01.001 https://doi.org/10.1016/j.physbeh.2007.11.050 Qu, D., Ludwig, D. S., Gammeltoft, S., Piper, M., Pelleymounter, M. A., Cullen, Sciolino, N. R., Hsiang, M., Mazzone, C. M., Wilson, L. R., Plummer, N. W., M. J., . . . Maratos-Flier, E. (1996). A role for melanin-concentrating Amin, J., . . . Jensen, P. (2022). Natural locus coeruleus dynamics during hormone in the central regulation of feeding behaviour. Nature, feeding. Sci Adv, 8(33), eabn9134. 380(6571), 243-247. https://doi.org/10.1038/380243a0 https://doi.org/10.1126/sciadv.abn9134 Rand, K. L., Otte, J. L., Flockhart, D., Hayes, D., Storniolo, A. M., Stearns, V., . . Sclafani, A., & Springer, D. (1976). Dietary obesity in adult rats: similarities to . Carpenter, J. S. (2011). Modeling hot flushes and quality of life in breast hypothalamic and human obesity syndromes. Physiol Behav, 17(3), 461- cancer survivors. Climacteric, 14(1), 171-180. 471. https://doi.org/10.1016/0031-9384(76)90109-8 https://doi.org/10.3109/13697131003717070 Seeley, R. J., van Dijk, G., Campfield, L. A., Smith, F. J., Burn, P., Nelligan, J. A., Richard, J. E., López-Ferreras, L., Anderberg, R. H., Olandersson, K., & Skibicka, . . . Schwartz, M. W. (1996). Intraventricular leptin reduces food intake K. P. (2017). Estradiol is a critical regulator of food-reward behavior. and body weight of lean rats but not obese Zucker rats. Horm Metab Res, Psychoneuroendocrinology, 78, 193-202. 28(12), 664-668. https://doi.org/10.1055/s-2007-979874 https://doi.org/10.1016/j.psyneuen.2017.01.014 Seeley, R. J., Yagaloff, K. A., Fisher, S. L., Burn, P., Thiele, T. E., van Dijk, G., . . Rinaman, L. (2010). Ascending projections from the caudal visceral nucleus of . Schwartz, M. W. (1997). Melanocortin receptors in leptin effects. Nature, the solitary tract to brain regions involved in food intake and energy 390(6658), 349. https://doi.org/10.1038/37016 expenditure. Brain Research, 1350, 18-34. Shi, H., Strader, A. D., Woods, S. C., & Seeley, R. J. (2007). Sexually dimorphic https://doi.org/10.1016/j.brainres.2010.03.059 responses to fat loss after caloric restriction or surgical lipectomy. Am J Rivera, H. M., & Eckel, L. A. (2010). Activation of Central, But Not Peripheral, Physiol Endocrinol Metab, 293(1), E316-326. Estrogen Receptors Is Necessary for Estradiol’s Anorexigenic Effect in https://doi.org/10.1152/ajpendo.00710.2006 Ovariectomized Rats. Endocrinology, 151(12), 5680-5688. Shughrue, P. J., Lane, M. V., & Merchenthaler, I. (1997). Comparative distribution https://doi.org/10.1210/en.2010-0731 of estrogen receptor-α and -β mRNA in the rat central nervous system. Journal of Comparative Neurology, 388(4), 507-525. 55 56 Ivana Maric Ivana Maric https://doi.org/https://doi.org/10.1002/(SICI)1096- orexin pathway. Endocrinology, 144(4), 1506-1512. 9861(19971201)388:4<507::AID-CNE1>3.0.CO;2-6 https://doi.org/10.1210/en.2002-220788 Sidlo, J., Zaviacic, M., & Kvasnicka, P. (1995). Night and day differences in the Valle, A., Català-Niell, A., Colom, B., García-Palmer, F. J., Oliver, J., & Roca, P. food-intake of laboratory rats Wistar and Koletsky strains. Bratisl Lek (2005). Sex-related differences in energy balance in response to caloric Listy, 96(12), 655-657. restriction. Am J Physiol Endocrinol Metab, 289(1), E15-22. Simerly, R. B., Chang, C., Muramatsu, M., & Swanson, L. W. (1990). Distribution https://doi.org/10.1152/ajpendo.00553.2004 of androgen and estrogen receptor mRNA-containing cells in the rat van Marken Lichtenbelt, W. D., Vanhommerig, J. W., Smulders, N. M., brain: an in situ hybridization study. J Comp Neurol, 294(1), 76-95. Drossaerts, J. M., Kemerink, G. J., Bouvy, N. D., . . . Teule, G. J. (2009). https://doi.org/10.1002/cne.902940107 Cold-activated brown adipose tissue in healthy men. New England Journal Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, of Medicine, 360(15), 1500-1508. M. M., Graham-Lorence, S., . . . et al. (1994). Aromatase cytochrome Walf, A. A., & Frye, C. A. (2010). Estradiol reduces anxiety- and depression-like P450, the enzyme responsible for estrogen biosynthesis. Endocr Rev, behavior of aged female mice. Physiol Behav, 99(2), 169-174. 15(3), 342-355. https://doi.org/10.1210/edrv-15-3-342 https://doi.org/10.1016/j.physbeh.2009.09.017 Sinclair, E. B., Hildebrandt, B. A., Culbert, K. M., Klump, K. L., & Sisk, C. L. Wallenius, K., Wallenius, V., Sunter, D., Dickson, S. L., & Jansson, J. O. (2002). (2017). Preliminary evidence of sex differences in behavioral and neural Intracerebroventricular interleukin-6 treatment decreases body fat in rats. responses to palatable food reward in rats. Physiology & Behavior, 176, 165- Biochem Biophys Res Commun, 293(1), 560-565. 173. https://doi.org/https://doi.org/10.1016/j.physbeh.2017.03.042 https://doi.org/10.1016/s0006-291x(02)00230-9 Skibicka, K. P., Hansson, C., Alvarez-Crespo, M., Friberg, P. A., & Dickson, S. L. Wallenius, V., Wallenius, K., Ahrén, B., Rudling, M., Carlsten, H., Dickson, S. L., (2011). Ghrelin directly targets the ventral tegmental area to increase food . . . Jansson, J. O. (2002). Interleukin-6-deficient mice develop mature- motivation. Neuroscience, 180, 129-137. onset obesity. Nat Med, 8(1), 75-79. https://doi.org/10.1038/nm0102- Smarr, B., & Kriegsfeld, L. J. (2022). Female mice exhibit less overall variance, 75 with a higher proportion of structured variance, than males at multiple Wang, C., & Xu, Y. (2019). Mechanisms for Sex Differences in Energy timescales of continuous body temperature and locomotive activity Homeostasis. J Mol Endocrinol, 62(2), R129-r143. records. Biol Sex Differ, 13(1), 41. https://doi.org/10.1186/s13293-022- https://doi.org/10.1530/jme-18-0165 00451-1 Wise, R. A. (2004). Dopamine and food reward: back to the elements. American Society, E. (2023). Some breast cancer treatments may limit effectiveness of weight loss Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 286(1), medications R13-R13. https://doi.org/10.1152/ajpregu.00590.2003 Stenlöf, K., Wernstedt, I., Fjällman, T., Wallenius, V., Wallenius, K., & Jansson, Wise, R. A., & Bozarth, M. A. (1984). Brain reward circuitry: four circuit elements J. O. (2003). Interleukin-6 levels in the central nervous system are "wired" in apparent series. Brain Res Bull, 12(2), 203-208. negatively correlated with fat mass in overweight/obese subjects. J Clin https://doi.org/10.1016/0361-9230(84)90190-4 Endocrinol Metab, 88(9), 4379-4383. https://doi.org/10.1210/jc.2002- Woitowich, N. C., Beery, A., & Woodruff, T. (2020). A 10-year follow-up study 021733 of sex inclusion in the biological sciences. eLife, 9, e56344. Takahashi, K., Hosoya, T., Onoe, K., Takashima, T., Tanaka, M., Ishii, A., . . . https://doi.org/10.7554/eLife.56344 Watanabe, Y. (2018). Association between aromatase in human brains Woods, S. C., Benoit, S. C., Clegg, D. J., & Seeley, R. J. (2004). Regulation of and personality traits. Scientific Reports, 8(1), 16841. energy homeostasis by peripheral signals. Best Practice & Research Clinical https://doi.org/10.1038/s41598-018-35065-4 Endocrinology & Metabolism, 18(4), 497-515. Timper, K., Denson, J. L., Steculorum, S. M., Heilinger, C., Engström-Ruud, L., https://doi.org/https://doi.org/10.1016/j.beem.2004.08.004 Wunderlich, C. M., . . . Brüning, J. C. (2017). IL-6 Improves Energy and Yang, B., Sanches-Padilla, J., Kondapalli, J., Morison, S. L., Delpire, E., Glucose Homeostasis in Obesity via Enhanced Central IL-6 trans- Awatramani, R., & Surmeier, D. J. (2021). Locus coeruleus anchors a Signaling. Cell Rep, 19(2), 267-280. trisynaptic circuit controlling fear-induced suppression of feeding. https://doi.org/10.1016/j.celrep.2017.03.043 Neuron, 109(5), 823-838.e826. Toshinai, K., Date, Y., Murakami, N., Shimada, M., Mondal, M. S., Shimbara, T., https://doi.org/10.1016/j.neuron.2020.12.023 . . . Nakazato, M. (2003). Ghrelin-induced food intake is mediated via the Yaswen, L., Diehl, N., Brennan, M. B., & Hochgeschwender, U. (1999). Obesity in the mouse model of pro-opiomelanocortin deficiency responds to 57 58 Ivana Maric Ivana Maric https://doi.org/https://doi.org/10.1002/(SICI)1096- orexin pathway. Endocrinology, 144(4), 1506-1512. 9861(19971201)388:4<507::AID-CNE1>3.0.CO;2-6 https://doi.org/10.1210/en.2002-220788 Sidlo, J., Zaviacic, M., & Kvasnicka, P. (1995). Night and day differences in the Valle, A., Català-Niell, A., Colom, B., García-Palmer, F. J., Oliver, J., & Roca, P. food-intake of laboratory rats Wistar and Koletsky strains. Bratisl Lek (2005). Sex-related differences in energy balance in response to caloric Listy, 96(12), 655-657. restriction. Am J Physiol Endocrinol Metab, 289(1), E15-22. Simerly, R. B., Chang, C., Muramatsu, M., & Swanson, L. W. (1990). Distribution https://doi.org/10.1152/ajpendo.00553.2004 of androgen and estrogen receptor mRNA-containing cells in the rat van Marken Lichtenbelt, W. D., Vanhommerig, J. W., Smulders, N. M., brain: an in situ hybridization study. J Comp Neurol, 294(1), 76-95. Drossaerts, J. M., Kemerink, G. J., Bouvy, N. D., . . . Teule, G. J. (2009). https://doi.org/10.1002/cne.902940107 Cold-activated brown adipose tissue in healthy men. New England Journal Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, of Medicine, 360(15), 1500-1508. M. M., Graham-Lorence, S., . . . et al. (1994). Aromatase cytochrome Walf, A. A., & Frye, C. A. (2010). Estradiol reduces anxiety- and depression-like P450, the enzyme responsible for estrogen biosynthesis. Endocr Rev, behavior of aged female mice. Physiol Behav, 99(2), 169-174. 15(3), 342-355. https://doi.org/10.1210/edrv-15-3-342 https://doi.org/10.1016/j.physbeh.2009.09.017 Sinclair, E. B., Hildebrandt, B. A., Culbert, K. M., Klump, K. L., & Sisk, C. L. Wallenius, K., Wallenius, V., Sunter, D., Dickson, S. L., & Jansson, J. O. (2002). (2017). Preliminary evidence of sex differences in behavioral and neural Intracerebroventricular interleukin-6 treatment decreases body fat in rats. responses to palatable food reward in rats. Physiology & Behavior, 176, 165- Biochem Biophys Res Commun, 293(1), 560-565. 173. https://doi.org/https://doi.org/10.1016/j.physbeh.2017.03.042 https://doi.org/10.1016/s0006-291x(02)00230-9 Skibicka, K. P., Hansson, C., Alvarez-Crespo, M., Friberg, P. A., & Dickson, S. L. Wallenius, V., Wallenius, K., Ahrén, B., Rudling, M., Carlsten, H., Dickson, S. L., (2011). Ghrelin directly targets the ventral tegmental area to increase food . . . Jansson, J. O. (2002). Interleukin-6-deficient mice develop mature- motivation. Neuroscience, 180, 129-137. onset obesity. Nat Med, 8(1), 75-79. https://doi.org/10.1038/nm0102- Smarr, B., & Kriegsfeld, L. J. (2022). Female mice exhibit less overall variance, 75 with a higher proportion of structured variance, than males at multiple Wang, C., & Xu, Y. (2019). Mechanisms for Sex Differences in Energy timescales of continuous body temperature and locomotive activity Homeostasis. J Mol Endocrinol, 62(2), R129-r143. records. Biol Sex Differ, 13(1), 41. https://doi.org/10.1186/s13293-022- https://doi.org/10.1530/jme-18-0165 00451-1 Wise, R. A. (2004). Dopamine and food reward: back to the elements. American Society, E. (2023). Some breast cancer treatments may limit effectiveness of weight loss Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 286(1), medications R13-R13. https://doi.org/10.1152/ajpregu.00590.2003 Stenlöf, K., Wernstedt, I., Fjällman, T., Wallenius, V., Wallenius, K., & Jansson, Wise, R. A., & Bozarth, M. A. (1984). Brain reward circuitry: four circuit elements J. O. (2003). Interleukin-6 levels in the central nervous system are "wired" in apparent series. Brain Res Bull, 12(2), 203-208. negatively correlated with fat mass in overweight/obese subjects. J Clin https://doi.org/10.1016/0361-9230(84)90190-4 Endocrinol Metab, 88(9), 4379-4383. https://doi.org/10.1210/jc.2002- Woitowich, N. C., Beery, A., & Woodruff, T. (2020). A 10-year follow-up study 021733 of sex inclusion in the biological sciences. eLife, 9, e56344. Takahashi, K., Hosoya, T., Onoe, K., Takashima, T., Tanaka, M., Ishii, A., . . . https://doi.org/10.7554/eLife.56344 Watanabe, Y. (2018). Association between aromatase in human brains Woods, S. C., Benoit, S. C., Clegg, D. J., & Seeley, R. J. (2004). Regulation of and personality traits. Scientific Reports, 8(1), 16841. energy homeostasis by peripheral signals. Best Practice & Research Clinical https://doi.org/10.1038/s41598-018-35065-4 Endocrinology & Metabolism, 18(4), 497-515. Timper, K., Denson, J. L., Steculorum, S. M., Heilinger, C., Engström-Ruud, L., https://doi.org/https://doi.org/10.1016/j.beem.2004.08.004 Wunderlich, C. M., . . . Brüning, J. C. (2017). IL-6 Improves Energy and Yang, B., Sanches-Padilla, J., Kondapalli, J., Morison, S. L., Delpire, E., Glucose Homeostasis in Obesity via Enhanced Central IL-6 trans- Awatramani, R., & Surmeier, D. J. (2021). Locus coeruleus anchors a Signaling. Cell Rep, 19(2), 267-280. trisynaptic circuit controlling fear-induced suppression of feeding. https://doi.org/10.1016/j.celrep.2017.03.043 Neuron, 109(5), 823-838.e826. Toshinai, K., Date, Y., Murakami, N., Shimada, M., Mondal, M. S., Shimbara, T., https://doi.org/10.1016/j.neuron.2020.12.023 . . . Nakazato, M. (2003). Ghrelin-induced food intake is mediated via the Yaswen, L., Diehl, N., Brennan, M. B., & Hochgeschwender, U. (1999). Obesity in the mouse model of pro-opiomelanocortin deficiency responds to 57 58 Ivana Maric peripheral melanocortin. Nat Med, 5(9), 1066-1070. https://doi.org/10.1038/12506 Yazdi, F. T., Clee, S. M., & Meyre, D. (2015). Obesity genetics in mouse and human: back and forth, and back again. PeerJ, 3, e856. https://doi.org/10.7717/peerj.856 Zandian, M., Ioakimidis, I., Bergh, C., Leon, M., & Södersten, P. (2011). A sex difference in the response to fasting. Physiol Behav, 103(5), 530-534. https://doi.org/10.1016/j.physbeh.2011.04.009 Zigman, J. M., Jones, J. E., Lee, C. E., Saper, C. B., & Elmquist, J. K. (2006). Expression of ghrelin receptor mRNA in the rat and the mouse brain. Journal of Comparative Neurology, 494(3), 528-548. 59